1. Ukuqonda kokuqala

Kweli nqanaba, kufuneka siqonde ezinye iikhonsepthi kunye nesigama, ukuze siphephe ukwenza iimpazamo phambi kwabantu abadala, njengale:

Umbuzo: Uthini umahluko phakathi kwe-RT-PCR, qPCR, i-PCR yexesha langempela, kunye ne-RT-PCR yexesha langempela?

Impendulo: I-RT-PCR yi-PCR eguqulelwe umva(i-reverse transcription PCR, RT-PCR), esetyenziswa ngokubanzi kwi-polymerase chain reaction (PCR).Kwi-RT-PCR, i-RNA strand iguqulelwa umva kwi-DNA ehambelanayo, ethi ke isetyenziswe njengethempleyithi yokukhulisa i-DNA yi-PCR.
Ixesha lokwenyani-PCR kunye qPCR(I-Quantitative Real-ltime-PCR) yinto efanayo, zombini i-PCR yobungakanani bexesha langempela, oku kuthetha ukuba umjikelo ngamnye we-PCR uneerekhodi zedatha yexesha langempela, ngoko ke inani leetemplates zokuqalisa linokuhlengahlengiswa uhlalutyo oluchanekileyo.

Nangona zombini i-PCR yexesha lokwenyani (ixesha lokwenyani le-fluorescent quantitative PCR) kunye ne-Reverse transcription PCR (reverse transcription PCR) ibonakala ifinyeziwe njenge-RT-PCR, indibano yamazwe ngamazwe yile: I-RT-PCR ibhekisa ngokukodwa kushicilelo olubuyela umva.I-PCR , I-PCR yexesha lokwenyani ifinyezwa ngokubanzi njenge-qPCR (ubungakanani bexesha lokwenyani PCR).

Kwaye i-RT-PCR yexesha langempela (RT-qPCR), Yi-PCR yokukhuphela ebuyela umva idityaniswe netekhnoloji yobungakanani befluorescent.: qala ufumane i-cDNA (RT) esuka kwi-RNA eguqulela umva, kwaye emva koko usebenzise i-PCR yexesha elililo lohlalutyo lobungakanani (qPCR).Iilabhoratri ezininzi zenza i-RT-qPCR, oko kukuthi, uphando kwi-RNA expression down-regulation, ke i-qPCR athetha ngayo wonke umntu elabhoratri ibhekisa kwi-RT-qPCR, kodwa ungalibali ukuba kusekho iimvavanyo ezininzi ze-DNA kwizicelo zeklinikhi.Uhlalutyo lobungakanani, olufana nentsholongwane ye-hepatitis B yokufumanisa i-HBV.

Umbuzo: Emva kokufunda i-PCR eninzi ye-fluorescent quantitative, kutheni i-fragment eyandisiweyo kufuneka ilawulwe phakathi koluhlu lwe-80-300bp?

Phendula: Ubude bolandelelwano lwe-gene nganye buhlukile, ezinye ziliqela kb, ezinye ngamakhulu e-bp, kodwa sifuna kuphela ubude bemveliso ukuba bube yi-80-300bp xa uyila iiprimers, ezimfutshane kakhulu okanye ezide kakhulu azifanelekanga ukufunyanwa kwe-fluorescent quantitative PCR.Iqhekeza lemveliso lifutshane kakhulu ukuba lingahlukaniswa ne-primer-dimer.Ubude be-primer-dimer malunga ne-30-40bp, kwaye kunzima ukuhlukanisa ukuba i-primer-dimer okanye imveliso ukuba ingaphantsi kwe-80bp.Ukuba i-fragment yemveliso ide kakhulu, idlula i-300bp, iya kukhokelela ngokulula ekusebenzeni okuphantsi kokukhulisa kwaye ayikwazi ukubona ngokufanelekileyo inani le-gene.

Umzekelo, xa ubala ukuba bangaphi abantu eklasini, kufuneka ubale ukuba mingaphi imilomo ekhoyo.Kuyafana naxa ubhaqa iijini, ufuna kuphela ukufumanisa ulandelelwano oluthile lwemfuza ukumela Ulandelelwano lonke luya kwenza.Ukuba ufuna ukubala abantu, kufuneka ubale imilomo neempumlo, iindlebe, neendondo, kwaye kulula ukwenza iimpazamo.

Ukwandisa, kuphando lwebhayoloji, kukho iimeko ezininzi zophando ukusuka kwindawo ukuya kwindawo, ngenxa yokuba ulandelelwano lwemfuza lwalo naluphi na uhlobo lude kakhulu, akuyomfuneko kwaye akunakwenzeka ukulinganisa zonke iziqwenga, ezifana nokulandelelana kwebhaktheriya ye-16S, okukukwenza ulandelelwano olulondolozayo lwee-Assays zebhaktheriya ukulinganisa inani elithile labantu beebhaktheriya.

Q: Bobuphi ubude obona bude boyilo lweprimer ye-qPCR?

Phendula: Ngokuqhelekileyo, ubude be-primer bu malunga ne-20-24bp, into engcono.Ngokuqinisekileyo, kufuneka sinikele ingqalelo kwixabiso le-TM ye-primer xa uyila i-primer, kuba oku kuhambelana nobushushu obuphezulu bokutshisa.Emva kovavanyo oluninzi, kubonakaliswe ukuba i-60 ° C lixabiso elingcono le-TM.Ukuba iqondo lobushushu le-anneal liphantsi kakhulu, liya kukhokelela ngokulula kulwandiso olungangqalanga.Ukuba ubushushu be-annealing buphezulu kakhulu, ukusebenza kakuhle kwe-amplification kuya kuba ngaphantsi, ukuphakama kwe-curve ye-amplification kuya kuqala kamva, kwaye ixabiso le-CT liya kulibaziseka.

Umbuzo: Yahluke njani indlela yedayi kwindlela yokuhlola?

Impendulo: Indlela yedayiEzinye iidayi zefluorescent, ezifana neSYBR Green Ⅰ, PicoGreen, BEBO, njl. njl., azikhuphi kukhanya ngokwazo, kodwa ziya kukhupha i-fluorescence emva kokubophelela kwigroove encinci ye-DNA ephindwe kabini.Ngoko ke, ekuqaleni kokusabela kwe-PCR, umatshini awukwazi ukubona isignali ye-fluorescent.Xa i-reaction ifikelela kwi-annealing-extension stage, i-strand ephindwe kabini ivuliwe, kwaye i-strand entsha yenziwe phantsi kwesenzo se-DNA polymerase, kwaye i-molecule ye-fluorescent ibophelela kwi-dsDNA encinci ye-groove.Njengoko inani lemijikelezo ye-PCR lisanda, iidayi ezininzi zidibene kunye ne-DNA ephindwe kabini, kwaye isignali ye-fluorescent nayo iphuculwe ngokuqhubekayo.Indlela yokudaya isetyenziswa kakhulu kuphando lwenzululwazi.
I-PS: Qaphela xa usenza uvavanyo, idayi kufuneka idibaniswe ne-DNA yomntu, qaphela ukuyijika ibe ngumntu we-fluorescent.

Indlela yokudaya (ekhohlo) Indlela yokuhlola (ekunene)
I-PS: Qaphela xa usenza uvavanyo, idayi kufuneka idibaniswe ne-DNA yomntu, qaphela ukuyijika ibe ngumntu we-fluorescent.

I-SYBR eluhlaza Ⅰ ibophelela kumqolo omncinci we-DNA

Indlela yokuhlolaI-Taqman probe yeyona nto isetyenziswa kakhulu kwi-hydrolysis probe.Kukho iqela le-fluorescent kwi-5′ ekupheleni kweprobe, ngokuqhelekileyo i-FAM, kwaye i-probe ngokwayo ilandelelanisa i-gene ekujoliswe kuyo.Kukho iqela lokucima i-fluorescent ekupheleni kwe-3′.Ngokomgaqo we-fluorescence resonance energy transfer (Förster resonance energy transfer, FRET), xa iqela lentatheli fluorescent (donor fluorescent molekyuli) kunye neqela lokucima i-fluorescent (i-acceptor fluorescent molekyuli) bayachulumancisa , ngelixa i-autofluorescence iyancipha.Ngoko ke, ekuqaleni kokuphendula kwe-PCR, xa i-probe ikhululekile kwaye ihambelana nenkqubo, iqela le-fluorescent yentatheli aliyi kukhupha i-fluorescence.Xa i-annealing, i-primer kunye ne-probe ibophelela kwi-template.Ngexesha lokwandiswa kwenqanaba, i-polymerase ngokuqhubekayo idibanisa amatyathanga amatsha.I-DNA polymerase ine-5'-3′ umsebenzi we-exonuclease.Xa ufikelela kwi-probe, i-polymerase ye-DNA iya ku-hydrolyze i-probe kwi-template, ihlukanise iqela le-fluorescent ye-reporter kwiqela le-fluorescent ye-quencher, kwaye ikhulule isignali ye-fluorescent.Ekubeni kukho ubudlelwane obunye phakathi kwe-probe kunye ne-template, indlela ye-probe iphezulu kunendlela yedayi ngokwemigaqo yokuchaneka kunye novakalelo lovavanyo.Indlela yokuhlola isetyenziswa kakhulu ekuxilongeni.

Umbuzo: Yintoni ubungakanani obupheleleyo?Yintoni iRelative Quantification?

Phendula: Ubungakanani obupheleleyo bubhekisa ekubalweni kwenombolo yokuqala yekopi yesampulu eza kuvavanywa yi-qPCR, njengokuba zingaphi iintsholongwane ze-HBV ezikwi-1ml yegazi.Isiphumo esifunyenwe ngomlinganiselo ohambelanayo lutshintsho kwixabiso lejene ekujoliswe kuyo kwisampulu ethile enxulumene nenye isampuli yereferensi, kwaye ukubonakaliswa kofuzo kulawulwa phezulu okanye phantsi.

Umbuzo: Ngaba isixa sokutsalwa kwe-RNA, ukubuyisela umva ukukhutshelwa ngokucokisekileyo, kunye nokwandiswa kwe-amplification kuchaphazela iziphumo zovavanyo?
Umbuzo: Ngaba ukugcinwa kwesampulu, ii-reagents zokukhutshwa, ii-reverse transcription reagents, kunye ne-transmitting consumables zichaphazela iziphumo zovavanyo?
Umbuzo: Yeyiphi indlela enokulungisa idatha yovavanyo?

Ngokumalunga nale miba, siya kuyichaza ngokweenkcukacha kumacandelo aphambili naphambili angezantsi.
2. Ulwazi oluphezulu

Ngokubhekiselele kwi-PCR yexesha lokwenyani le-fluorescent quantitative, kufuneka siqonde ubunyani bokuba amawaka amaphepha ophando lwesayensi apapashwa minyaka le, phakathi kwawo iteknoloji ye-PCR yobungakanani befluorescent ayilo nani lincinci.

Ukuba akukho mgangatho uqhelekileyo wokulinganisa umfuniselo we-PCR wobungakanani befluorescent, iziphumo zinokwahluka kakhulu.Kwimfuza efanayo yohlobo olufanayo, kunye nendlela efanayo yokucubungula, iziphumo zokufumanisa nazo ziya kwahluka ngokubanzi, kwaye kuya kuba nzima ukuba abafika emva kwexesha baphinde iziphumo ezifanayo.Wena Akukho mntu waziyo ukuba yeyiphi elungileyo nengalunganga.

Ngaba oku kuthetha ukuba i-fluorescent quantitative PCR yitekhnoloji yokukopela okanye iteknoloji engathembekanga?Hayi, kungenxa yokuba i-fluorescent quantitative PCR inovakalelo ngakumbi kwaye ichaneke ngakumbi, kwaye umsebenzi omncinci ongalunganga uya kuvelisa iziphumo ezichasene ngokupheleleyo.Ilahleko encinci ikwiwaka leekhilomitha kude.Umbhali wenqaku unokuxhatshazwa ngokuphindaphindiweyo ngabahlalutyi.Kwangaxeshanye, abaphononongi bejenali nabo kunzima ukukhetha kwiziphumo ezahlukeneyo zovavanyo.

Lilonke, likhomba ukunqongophala kwemvumelwano kwimifuniselo yePCR yexesha lokwenyani.Ukuza kuthi ga ngoku, izazinzulu eziphezulu kwishishini zaqala ukwenza imigangatho,ifuna abaxhasi ukuba babonelele ngovavanyo oluyimfuneko kunye neenkcukacha zokucubungula idatha (kuquka idatha efunekayo) kwinqaku ukuhlangabezana nale migangatho.

Ababuyekezi banokugweba umgangatho wovavanyo ngokufunda ezi nkcukacha;abafundi bexesha elizayo banokusebenzisa oku ukuphinda umfuniselo okanye ukuphucula umfuniselo.Emva koko iziphumo zovavanyo ezifunyenwe ngolu hlobo zigcwele ulwazi, umgangatho ophezulu, kunye nokusebenziseka.

I-MIBBI (Ubuncinci boLwazi lweBiological and Biomedical Investigations -http://www.mibbi.org) kwabakho.I-MIBBI yiprojekthi ebonelela ngemigangatho yovavanyo.Ipapashwa kwindalo.Le projekthi ijolise kwiimvavanyo ezahlukeneyo zebhayoloji, kubandakanya ibhayoloji yeseli, iMicroarray, i-qPCR esiza kuyixoxa ngoku, njl. njl., kwaye ibonelela ngohlobo ngalunye lovavanyo xa kufakwa imibhalo-ngqangi.Olo lwazi kufuneka lunikezelwe ngalo lonke ixesha.

Kwiprojekthi ye-MIBBI, kukho amanqaku amabini anxulumene ne-fluorescent quantitative PCR, angala.:
·RDML (Ixesha langempela le-PCR yeNkcukacha yoLwabiwo lweDatha) - ulwimi olucwangcisiweyo kunye nesikhokelo sokunika ingxelo kwidatha ye-PCR yexesha langempela;
·MIQE (Ubuncinci boLwazi lokuPapashwa koMlingo we-PCR weXesha eliNina) – ubuncinci bolwazi lokupapasha amanqaku amalunga nexesha lokwenyani lovavanyo lwe-PCR.
Okokuqala, makhe sithethe nge-RDML, inkcazo yesigama.

Ukuba akukho nkcazo eqhelekileyo kuyo yonke into, akunakwenzeka ukuqhubeka nengxoxo, yingakho inkcazo yamagama ibaluleke kakhulu kuviwo.
Isigama esisetyenziswe kuvavanyo lwe-fluorescent quantitative PCR lubandakanya lo mxholo ulandelayo.U-QIAGEN wenze esona sishwankathelo esihle kuthi.Ezi zilandelayo zomile zonkeiimpahla .

Igophe lokukhulisa
Ijika le-amplification libhekisela kwi-curve eyenziwe ngexesha lenkqubo ye-PCR, kunye nenombolo yomjikelezo njenge-abscissa kunye ne-real-time fluorescence intensity ngexesha lokuphendula njenge-ordinate.

Igophe eligqwesileyo lokukhulisa kufuneka libe nezi mpawu zilandelayo: isiseko sisicaba okanye sinciphe kancinci, kwaye akukho ndlela icacileyo yokuya phezulu;indawo ye-inflection ye-curve icacile, kwaye i-slope yesigaba se-exponential ihambelana nokusebenza kakuhle kokukhulisa.Okukhona umthambeka uphezulu, kokukhona ukukhulisa ukusebenza kakuhle kokukhulisa;i-curve ye-amplification jikelele I-parallelism ilungile, ebonisa ukuba ukusebenza kakuhle kwe-tube nganye kuyafana;Isigaba sokwandisa igophe lokwandiswa kweesampulu ezisezantsi zicacile.

Isiseko (Isiseko)
Isiseko yinqanaba lengxolo yomjikelo wokuqala, ngokuqhelekileyo kulinganiswa phakathi komjikelezo we-3 kunye ne-15, kuba ukunyuka kwexabiso le-fluorescence okubangelwa yimveliso yokukhulisa ayikwazi ukufunyanwa ngeli xesha.Inani lemijikelezo esetyenziselwa ukubala isiseko sinokuhlukahluka kwaye kunokufuneka kuncitshiswe ukuba iimali zetemplate eziphezulu zisetyenzisiwe okanye ukuba inqanaba lokubonakaliswa kwejene elijoliswe kuyo liphezulu.

Ukumisela isiseko kufuna ukujonga idatha ye-fluorescence ukusuka kwi-curve yokukhulisa umgca.Isiseko simiselwe ukwenzela ukuba ukukhula kwe-curve yokukhulisa iqale ngenani lomjikelo omkhulu kunomjikelo wesiseko inani eliphezulu.Iziseko kufuneka zisekwe ngamnye kulandelelwano lweethagethi nganye.Amaxabiso e-fluorescence aphakathi afunyanwe kwimijikelezo yokuqala kufuneka asuswe kumaxabiso e-fluorescence afunyenwe kwiimveliso ezikhulisiwe.Iinguqulelo zamva nje ezahlukeneyo zesoftware yePCR yeXesha lokwenyani zivumela ulungiselelo oluzenzekelayo lwesiseko sesampulu nganye.

Ngexesha lemijikelo embalwa yokuqala ye-PCR yokusabela kokukhulisa, isignali ye-fluorescence ayitshintshi kakhulu.Ukusondela kumgca othe ngqo kubizwa ngokuba sisiseko, kodwa ukuba sijonga ngokusondeleyo kwimijikelo embalwa yokuqala, sibona ukuba ngaphakathi kwesiseko kwenzeka ntoni kulo mfanekiso ungezantsi.

Imvelaphi ibhekisa kwi
ixabiso le-fluorescence elingangcalulinga kwindlela yokusabela .Umzekelo: ukucima i-fluorescence engasebenziyo;okanye inani elikhulu leetemplates eziphindwe kabini zeDNA ngenxa yokusetyenziswa kweSYBR Green.Amacandelo angasemva ophawu asuswa ngokwezibalo yi-algorithm yesoftware ye-PCR yexesha lokwenyani.

Umqondiso weNtatheli
Umqondiso weNtatheli ubhekisa kumqondiso wefluorescent oveliswe yi-SYBR Green okanye i-fluorescent ephawulwe ngolandelelwano-iprobes ethile ngexesha le-PCR yeXesha lokwenyani.

Umqondiso weNtatheli oQhelekileyo (RN)
I-RN ibhekiselele kukuqina kwe-fluorescence yedayi yentatheli eyahlulwe ngamandla e-fluorescence yedayi yereferensi yokwenziwa elinganiswe kumjikelo ngamnye.

Passive Reference Idayi
Kwezinye iiPCR zeXesha lokwenyani,Idayi ye-fluorescent i-ROX isetyenziswa njengesalathiso sangaphakathi sokulungelelanisa isignali ye-fluorescent.Ilungisa iiyantlukwano ngenxa yokungachaneki kwemibhobho, indawo yokuma kakuhle, kunye nokuguquguquka kwe-fluorescence ngokufanelekileyo.

Umda we-fluorescence (umda)
yahlengahlengiswa ngaphezulu kwexabiso elingasemva kwaye ngokubalulekileyo ngaphantsi kwexabiso leplateau legophe lokukhulisa.Imele ilale kwindawo yomgca wegophe lokukhulisa, emele uluhlu lwe-log-linear yokufunyanwa kwe-PCR.I-Thresholds kufuneka imiselwe kwi-log-amplification curve view ukuze i-log-linear phase ye-PCR ibonakale lula.Ukuba kukho iijini ekujoliswe kuzo ezininzi kwi-Real-Time PCR, umda kufuneka umiselwe kwithagethi nganye .Ngokuqhelekileyo, umqondiso we-fluorescence wemijikelo yokuqala ye-15 ye-PCR reaction isetyenziswa njengophawu lwangasemva lwe-fluorescence, kwaye i-fluorescence threshold yi-10 amaxesha okutenxa okusemgangathweni komqondiso we-fluorescence wokuqala we-3 ukuya kwi-15 ye-PCR, kwaye i-fluorescence threshold ibekwe kwi-PCR ye-amponplential.Ngokubanzi, isixhobo ngasinye sine-fluorescence threshold ebekwe phambi kokusetyenziswa.

I-Cycle Threshold (CT) okanye indawo yokunqumla (CP)
Umjikelo apho ijiko lokukhulisa linqumla umda (oko kukuthi, indawo apho ukubonwa kwe-fluorescence kwanda kakhulu).I-CT ingaba yiqhezu kwaye inani lokuqalisa itemplate lingabalwa.Ixabiso le-CT limele inani lemijikelo efunyenweyo xa isignali ye-fluorescent kwityhubhu nganye yokusabela ye-PCR ifikelela kumda omiselweyo.Kukho ubudlelwane bomgca phakathi kwexabiso le-CT letemplate nganye kunye nelogarithm yenombolo yokuqala yekopi yetemplate,aphezulu inani lekopi yokuqala, ixabiso elincinci le-CT, kwaye ngokuphambene.Ijika eliqhelekileyo linokwenziwa ngokusebenzisa umgangatho kunye nenombolo yekopi yokuqala eyaziwayo, apho i-abscissa ibonisa ixabiso le-CT, kwaye i-ordinate imele i-logarithm yenombolo yokuqala yekopi.Ngoko ke, ngokude nje ixabiso le-CT yesampuli engaziwayo ifunyenwe, inombolo yokuqala yekopi yesampuli ingabalwa kwi-curve standard.

ΔCT ixabiso
ΔCT ixabiso liyachazaUmahluko phakathi kwejini ekujoliswe kuyo kunye nexabiso le-CT yereferensi ehambelanayo, njengejini yokugcina indlu, kwaye isetyenziselwa ukulungelelanisa isixa setemplate esisetyenzisiweyo:
⇒ΔCT = CT (igene ekujoliswe kuyo) - CT (i-endogenous reference gene)

ΔΔCT Ixabiso
Ixabiso ΔΔCT lichaza umahluko phakathi kwexabiso ΔΔCT lesampulu yomdla (umzekelo, iiseli ezivuselelweyo) kunye nentsingiselo ΔΔCT yexabiso lereferensi (umzekelo, iiseli ezingavuselelwanga).Isampulu yesalathiso ikwabizwa ngokuba yisampulu yohlengahlengiso kwaye zonke ezinye iisampulu ziqhelekile koku ukuze kulinganiswe umyinge:
⇒ΔΔCT = i-avareji ΔCT (isampulu yomdla) - i-avareji ΔCT (isampulu yereferensi)

Ijene yereferensi engapheliyo (imizila yereferensi engapheliyo)
Amanqanaba okuchazwa kwejene yereferensi engapheliyo, efana nejini yokugcina indlu (ijene yokugcina indlu), awahlukanga phakathi kweesampulu.Ukuthelekisa amaxabiso e-CT yejini yereferensi kuhlobo lofuzo ekujoliswe kulo kuvumela inqanaba lokubonisa lejini ekujoliswe kuyo ukuba ibe yesiqhelo kwisixa segalelo le-RNA okanye i-cDNA (bona icandelo kumaxabiso e-ΔCT apha ngasentla).

Ufuzo lwangaphakathi lwereferensi oluchanekileyoUkutshatyalaliswa okunokwenzeka kwe-RNA okanye ubukho be-enzyme inhibitors kwiisampuli ze-RNA, kunye nokwahluka komxholo we-RNA, ukubuyisela ukuguqulwa kokubhala kakuhle, ukubuyiswa kwe-nucleic acid, kunye nokuphathwa kwesampuli.Ukukhetha eyona gene(s) yereferensi, silungise ialgorithm ukuvumela ukhetho lwayo lwereferensi elolona luxhomekeke kulungiselelo lovavanyo.

Ulawulo lwangaphakathi
Ulandelelwano lolawulo olwandiswa ngendlela efanayo njengolandelelwano lwethagethi kwaye luphononongwe ngeprobe eyahlukileyo (okt, ukwenza i-duplex PCR).Ulawulo lwangaphakathi luhlala lusetyenziselwa ukulawula ulwandiso olungaphumeleliyo, njengaxa ulandelelwano ekujoliswe kulo alubonwa.
Isampulu yokulinganisa
Isampulu yereferensi (umzekelo, i-RNA esulungekileyo ukusuka kumgca weseli okanye izicubu) esetyenziswa kumlinganiselo onxulumeneyo ukuthelekisa zonke ezinye iisampuli ukumisela inqanaba lokubonisa elizalanayo lemfuza.Isampulu yokulinganisa ingaba nayiphi na isampuli, kodwa idla ngokuba lulawulo (umzekelo, isampuli engaphendulwanga okanye isampuli ukusuka kwixesha le-zero yovavanyo).

Ulawulo oluhle
sebenzisa iimpendulo zolawulo ngeizixa ezaziwayo template.Ulawulo oluhle luhlala lusetyenziselwa ukujonga ukuba iseti yeprimer okanye iseti ye-primer-probe isebenza ngokufanelekileyo kwaye impendulo imiselwe ngokuchanekileyo.

Akukho Template Control (NTC)
Ukusabela kokulawula okuqulethe zonke iinxalenye eziyimfuneko ze-amplification reaction ngaphandle kwetemplate, edla ngokutshintshwa ngamanzi.Ukusetyenziswa kwe-NTC kunokufumana ungcoliseko olubangelwa ungcoliseko lwe-reagent okanye i-DNA yangaphandle, ngaloo ndlela iqinisekisa ubunyani kunye nokuthembeka kwedatha yokufumanisa.Ukwandiswa kolawulo lwe-NTC lubonisa ukosuleleka.

Akukho lawulo lweRT (NRT)
Inkqubo yokutsalwa kwe-RNA isenokuqulatha intsalela ye-DNA ye-genomic, eyingozi kakhulu kwaye ingunobangela ochaphazela umgangatho wedatha kunye notshaba lwendalo lwe-qPCR, ngoko xa kuyilwa imifuniselo, kufuneka iyilwe ukukhulisa ubhaqo lwe-RNA kuphela.Kukho iindlela ezimbini, enye kukuyila ii-primers kwii-introns, enye kukukhupha ngokupheleleyo i-DNA, eyona nto ingcono, eya kuxoxwa ngayo kamva.Ulawulo lwe-NTR sisipili somlingo sokubona ungcoliseko lweDNA.Ukuba kukho ukukhulisa, kuthetha ukuba kukho ungcoliseko.

Imigangatho
Imigangatho yiisampulu zoxinaniso olwaziwayo okanye inombolo yekopi ezisetyenziselwa ukwakha igophe eliqhelekileyo.Ukuze kuqinisekiswe ukuzinza komgangatho, i-gene fragment idla ngokudibanisa kwi-plasmid kwaye isetyenziswe njengomgangatho.

Igophe elisemgangathweni
ngokuqhelekileyo ihlanjululwe ubuncinane kwi-5 i-concentration gradients kunye nemveliso esemgangathweni ngokwe-double ratio, kwaye amanqaku e-5 atsalwa kwii-coordinates zexabiso le-CT kunye nenombolo yokukopisha, kwaye amanqaku axhunyiwe ukwenza umgca wokuvelisa i-curve standard.Kwigophe eliqhelekileyo ngalinye, ukufaneleka kwayo kufuneka kuhlolwe.Ixabiso le-slope liwela phakathi kwe--3.3 kunye ne-3.8, kwaye ugxininiso ngalunye lwenziwa kathathu.Amanqaku ahluke kakhulu kwamanye afanele alahlwe.Ixabiso le-CT lesampula eliza kuvavanywa lingeniswa kwi-curve esemgangathweni, kwaye inqanaba lokuvakalisa isampula eliza kuvavanywa lingabalwa.

Ixabiso le-CT lesampula eliza kuvavanywa lingeniswa kwi-curve esemgangathweni, kwaye inombolo yokuqala yekopi yesampuli ukuba ivavanywe ingabalwa.

Ukusebenza kakuhle kunye ne-Slope
I-slope ye-curve esemgangathweni ibonisa ukusebenza kakuhle kwe-PCR yexesha langempela.
·I-slope ye--3.322 ibonisa ukuba i-PCR yokukhulisa ukusebenza kakuhle yi-1, okanye i-100% esebenzayo, kwaye inani lemveliso ye-PCR liphindwe kabini kumjikelo ngamnye.
·Ithambeka elingaphantsi kwe--3.322 (umz., –3.8) libonisa ukusebenza kakuhle kwePCR
·I-slope enkulu kune--3.322 (umz., -3.0) ibonisa ukuba ukusebenza kwe-PCR kubonakala kukhulu kune-100%, into enomdla, ingaba umjikelo omnye we-PCR unokuvelisa njani ngaphezu kokuphindwe kabini kwemveliso eyandisiweyo?Le meko yenzeka kwisigaba esingeyo-linear ye-reaction ye-PCR, oko kukuthi, kukho isixa esikhulu sokwandiswa kwe-non-specific.

ijika elinyibilikayo
Emva kokuba ulwandiso lwe-qPCR lugqityiwe, imveliso yePCR iyafudunyezwa.Njengoko iqondo lokushisa liphakama, imveliso yokukhulisa i-double-stranded iyancibilika, okukhokelela ekunciphiseni kokuqina kwe-fluorescence.Xa ubushushu obuthile (Tm) bufikelelwe, inani elikhulu leemveliso liya kunyibilika.I-Fluorescence ihla ngokukhawuleza.Iimveliso zePCR ezahlukeneyo zinexabiso leTm ezahlukeneyo kunye namaqondo obushushu anyibilikayo ahlukeneyo, ukuze kuchongwe ukucaciswa kwePCR.

Igophe elinyibilikayo (igophe eliphuma kwigophe)
Igophe lokunyibilika lithathwe ukwenza imephu ephezulu, enokuthi ibonise ngokubonakalayo imeko yamaqhekeza emveliso yePCR.Ekubeni iqondo lokushisa elinyibilikayo lixabiso le-Tm leqhekeza le-DNA, ezinye iiparitha ezichaphazela ixabiso le-Tm leqhekeza le-DNA linokugwetywa, njengobungakanani beqhekeza, umxholo we-GC, njl. Ngokuqhelekileyo, ngokwemigaqo yethu yokuyila i-primer,ubude bemveliso eyandisiweyo ikuluhlu lwe-80-300bp, ngoko iqondo lokushisa lokunyibilika kufuneka libe phakathi kwe-80 ° C kunye ne-90 ° C.

Ukutolikwa kwegophe lokunyibilika: Ukuba incopho ephambili kuphela ibonakala phakathi kwe-80 ° C-90 ° C, ithetha ukuba i-fluorescent quantitative PCR igqibelele;ukuba incopho ephambili ibonakala phakathi kwe-80 ° C-90 ° C kunye neencopho ezixubileyo zivela ngaphantsi kwe-80 ° C, i-primer dimer iqwalaselwa ngokusisiseko.Ungazama ukwandisa iqondo lobushushu le-anneal ukuyisombulula;ukuba incopho ephambili ibonakala phakathi kwe-80 ° C-90 ° C, kwaye i-miscellaneous peak ibonakala kwakhona xa iqondo lokushisa likhuphuka, kuthathwa ngokusisiseko ukuba kukho ukungcoliswa kwe-DNA, kwaye i-DNA kufuneka isuswe kwinqanaba lokuqala lovavanyo.

Ewe, kusekho ezinye iimeko ezingaqhelekanga, eziya kucazululwa nganye nganye ngezantsi.
3. Ulwazi oluphezulu

Ukwenza i-qPCR, kufuneka ndithi MIQE,Ubuncinane boLwaziyoPapasho lweUbungakananiI-PCR yexesha lokwenyaniIimvavanyo-ulwazi oluncinci lokupapasha amanqaku malunga ne-real - time quantitative PCRimifuniselo .Ukuze kube lula ukuqonda komntu wonke, siya kuwenza lula umxholo ophambili.

Unokukhangela umbhalo wokuqala we-MIQE kwi-Intanethi, kwaye eyona nto ibalulekileyo kukuba ibekauluhlu lokutshekisha idatha ekufuneka ibonelelwe xa upapasha inqaku .

Ababuyekezi banokugweba umgangatho wovavanyo ngokufunda ezi nkcukacha;abafundi bexesha elizayo banokusebenzisa oku ukuphinda okanye ukuphucula umfuniselo.
Kubalulekile ukuqaphela ukuba kolu luhlu, ukubaluleka koluhlu ngalunye kuphawulwe ngo-E okanye ngo-D ngokulandelelanayo.Ingaba ithetha ntoni?E: ulwazi oluyimfuneko (kufuneka lungeniswe);D: ulwazi olunqwenelekayo (bonelela kangangoko kunokwenzeka).

I-MIQE (1)—Uyilo loMfuniselo
Uninzi lwe-scumbags olugqibe ukuzikhusela emva kokugqiba izifundo zabo zesidanga aluyi kukwazi ukuyila umfuniselo ngokuzimeleyo, bavule iincwadi zabo zokubhalela, kwaye benze oko utitshala abaxelela ukuba bakwenze.Ngenxa yoko, uyilo lovavanyo aluzange lube ngqongqo, kwaye isebe lomhleli wephephancwadi lathi lifuna ukwenza lo mfanekiso kunye naloo mfanekiso, ngoko bakwenza bedidekile.Zenziwa ngolu hlobo ke iingcoli!

Kufuphi nekhaya, umgaqo wokuqala wovavanyo kukumiselaubungqongqo bengqiqo yovavanyo.Eyona nto isisiseko luyilo lokulinga, kwaye eyona nto ibalulekileyo malunga noyilo lokulinga yindlela yokuseta isampuli ekujoliswe kuyo, isampulu yesalathiso (ulawulo), kunye nenani lokuphindaphinda, ukuze idatha yovavanyo ibhekiselele, ithelekiseke, kwaye ikholise.

Isampulu ekujoliswe kuyoibhekisa kwisampulu efuna ukuba sibhaqe ijini ekujoliswe kuyo emva konyango oluthile.Isampulu yereferensiyisampuli ngaphandle kwalo naluphi na unyango, olusoloko lubizwa ngokuba luhlobo lwasendle kwibhayoloji.

Iikopi zovavanyozibaluleke kakhulu.Ngokuqhelekileyo, inani leekopi ezicengayo kufuneka zibe ngaphezu kwesithathu.Kuyimfuneko ukwahlula ukuba yintoni uphindaphindo lwebhayoloji kwaye yintoni uphindaphindo lobugcisa.

Biological Replicates: Uvavanyo olufanayo lokuqinisekisa olwenziwe ngezinto ezahlukeneyo (ixesha, izityalo, iibhetshi, iipleyiti zokusabela).

Uphindaphindo lwebhayoloji
Makhe sithathe unyango lwepesticide lwepepile njengomzekelo.Sifuna ukutshiza izibulali zinambuzane kwizityalo ezithathu ze-ABC, emva koko izityalo ezithathu ze-ABC ziziphindaphindo ezintathu zebhayoloji, kwaye ziluvavanyo olufanayo lokuqinisekisa olwenziwa ngezinto ezahlukeneyo.Kodwa njengovavanyo, ulawulo luyafuneka ngokuqinisekileyo, ngoko ke sinokutshiza elinye lamasebe esityalo A ukwenza iqela lovavanyo lwesityalo A, kwaye singatshizi amanye amasebe esityalo A ukwenza iqela lolawulo.Yenza okufanayo ku-B no-C.

IiReplicates zobuGcisa (IiReplicates zoBugcisa): Luvavanyo oluphindaphindiweyo oluyilelwe ukuphepha iimpazamo ezibangelwa kukusebenza, eneneni ngumngxuma ophindwe kabini oqukwe kwinto enye.Zombini unyango kunye nolawulo kufuneka lube nezicwangciso eziphindaphindayo (ubuncinci ezithathu) zejini ekujoliswe kuzo kunye nejene yereferensi yangaphakathi.

Ukuphindaphinda kobugcisa
Thatha ipepile ephathwe ngezibulali zinambuzane njengomzekelo kwakhona.Kwiqela lovavanyo lwesityalo esingu-A, senze imingxunya emithathu ye-PCR ka-1, 2, kunye no-3 kwijeni ekujoliswe kuyo kunye nejeni yereferensi yangaphakathi ngokulandelelana, ukuze sithathe umndilili emva kokubhaqwa.Kulawulo lwesityalo A Amaqela nawo aphathwa ngendlela efanayo.Ngokufanayo, yenza unyango olufanayo kwizityalo ze-B kunye ne-C.Oku kuphindaphindwa kobugcisa.

Kufanelekile ukuphawula okointo engena kwizibalo kukuphindwa kwebhayoloji, kwaye ukuphindaphinda kobugcisa kukuvavanya ukuba kukho naziphi na iziganeko ezingalindelekanga kwinkqubo yovavanyo, ukuze wenze iziphumo zovavanyo zithembeke, oko kukuthi, ukuphepha iimpazamo ngokuthatha umyinge wazo njengoko sihlala sithetha.

Ulawulo olubi—i-NTC kunye ne-NRT
NTC (Akukho-Template Control), ulawulo olungenayo itemplate, lusetyenziselwa ukuqinisekisa ukuba izinto zovavanyo zingcolisekile.Ngokuqhelekileyo, amanzi asetyenziswa njenge template.Ukuba kukho ukusabela kwe-fluorescent, kubonisa ukuba ukungcola kwe-nucleic acid kwenzeke kwibhubhoratri.

Olu ngcoliseko luvela: kumanzi angcolileyo, ii-reagents ezingafanelekanga eziqulethe i-DNA engapheliyo, ungcoliseko lwe-primer, ungcoliseko lwezixhobo zelabhoratri, ungcoliseko lwe-aerosol, njl., kufuneka kusetyenziswe i-RNase scavengers kunye ne-RNase inhibitors.Ungcoliseko lwe-aerosol yeyona nto inzima ukuyifumana.Khawube nomfanekiso-ngqondweni welabhoratri yakho ifana nomsi, eneeasidi ezahlukeneyo zenucleic ezixhonywe emoyeni.

NRT (No-Reverse Transcriptase), ulawulo olungenayo i-reverse transcription, yi-RNA engaguqukiyo njengolawulo olubi, olululawulo lwentsalela ye-gDNA.

Xa usenza i-gene expression, isixa se-RNA sichongwa ngokufumanisa isixa se-cDNA emva kokukhutshelwa umva.Ukuba kukho intsalela ye-gDNA xa i-RNA ihlanjululwa, iya kubangela iimpazamo kwiziphumo zovavanyo, kuba iziphumo ezifunyenweyo ziyi-gDNA kunye ne-cDNA.Kwinqanaba elidityanisiweyo, hayi nje i-cDNA, i-gDNA kufuneka isuswe ngokupheleleyo ngexesha lokutsalwa kwe-RNA.

I-MIQE (2) -ingcaciso yesampuli
Ulwazi olubizwa ngokuba lulwazi lwesampulu luthetha ukuba xa sipapasha inqaku malunga ne-qPCR, kufuneka sichaze ulwazi lwesampulu ngokucacileyo, oluyinxalenye ebalulekileyo yenqaku.Ngokufanayo, xa siqhuba iisampulu, kufuneka silawule imisebenzi yethu ukuqinisekisa ukunyaniseka kweesampuli.

Inkcazo yesampulu sisiphumo kuphela, kwaye kufuneka sinikele ingqalelo ngakumbi kwizinto ezithathiweyo ngexesha lonke lovavanyo.

Ukukhethwa kwezinto zokulinga
Iisampuli zegazi - khetha igazi elitsha, akukho ngaphezu kweeyure ze-4.Iisampulu zeseli - khetha ukuqokelela iiseli ezintsha ngexesha lokukhula ngamandla.Izicubu zezilwanyana-Khetha izicubu ezitsha, ezikhula ngamandla.Izicubu zezityalo - Khetha izicubu ezitsha, ezincinci.

Umele ukuba uqaphele ukuba kukho igama eliphambili kwezi zivakalisi zimbalwa: fresh .
Kwezi sampuli zingasentla, eyona khithi ingcono, ingabizi kakhulu, kwaye izinzile kwimarike yikhithi ye-Foregene, enokuthi ikhuphe ngokukhawuleza nangokulula i-DNA kunye ne-RNA.

Blood DNA Mini Kit

ISeli iyonke yeRNA Isolation Kit

Animal Total RNA Isolation Kit

Plant Total RNA Isolation Kit

Plant Total RNA Isolation Kit Plus

Plant DNA Isolation Kit

Ukugcinwa kwezinto zokulinga
Ngokuqhelekileyo, asicebisi ukugcina iisampulu, ukuba iimeko zivuma.Nangona kunjalo, kukho abahlobo abaninzi abangakwaziyo ukuqhuba iimvavanyo ngoko nangoko emva kokuthatha iisampulu, kwaye abanye bade bafune ukuthwala iitanki zenitrogen ezimanzi ukuya ebaleni ukuze kwenziwe isampulu.

Kulolu hlobo lomhlobo osebenza nzima, ndingatsho kuphela ukuba awuyiqondi i-reagent consumables.Ngoku iinkampani ezininzi ezisebenzisekayo ezisebenzisekayo zivelisa ii-reagents ezinokugcina iisampulu ze-RNA kwindawo yobushushu begumbi, kwaye unokukhetha ukuzisebenzisa.Indlela yogcino lwesiqhelo lulwelo ugcino nitrogen, usebenzisa itanki nitrogen engamanzi encinane ukuba kulula ukuthwala.Emva kokubuyisela isampuli kwibhubhoratri, yigcine kwi -80 ° C efrijini.

Kuvavanyo olubandakanya i-RNA, umgaqo wamagama amathandathu kufuneka ulandelwe:ubushushu obuphantsi, akukho enzymes;kwayengokukhawuleza .

Ingcamango yobushushu obuphantsi kulula ukuyiqonda;ngaphandle kwee-enzymes, i-RNase ikuyo yonke indawo kwihlabathi esiphila kulo (kungenjalo ubuya kubulawa yi-HIV), ngoko indlela yokuphepha i-RNase xa usenza imifuniselo yingcamango ebaluleke kakhulu;ngokukhawuleza,Akukho Kung Fu kwihlabathi elingenakophulwa, isantya kuphela asinakophulwa.

Ke ngoko, ukuba lifutshane ixesha lokutsalwa, kokukhona ikhithi ingcono.KutheniI-ForegeneIkhithi igxininisa isantya, kuba bayazi kakuhle.

I-PS: Amanye amantombazana enza imifuniselo ngononophelo olukhulu, kodwa awalunganga njenge-slam dunk emva kweminyaka eliqela yomsebenzi.Bavakalelwa kukuba uThixo akanabulungisa, uyabakhalazela abanye yaye ukhangela ubomi.Enyanisweni, wayengayiqondi.Akazange ayikhusele kakuhle i-RNA, kwaye umdlali we-slam dunk wayenomdla.Xa wayesenza umfuniselo, wacinga ukuba uza kugqibezela i-slam dunk ngokuphindwe kathathu, kahlanu kunye nezahlulo ezimbini, kodwa ulwenze kakuhle olo vavanyo.

Phawula: Kancinci, amathuba amaninzi okuhlasela kwe-RNase.Uziqeqeshe njani ukuba ukhawuleze?Akukho ndlela, ziqhelanise ngakumbi.

Kwimifuniselo eyahlukeneyo kunye neesampulu ezahlukeneyo, kusafuneka ukufunda uncwadi oluninzi kwaye ukhethe indlela efanelekileyo yokusetyenzwa.Ukuqokelela isampula kunye nenkqubo yokugcina, i-MIQE idinga ukuba kufuneka ibhalwe ngokucacileyo kwiphepha, ukwenzela ukuba ababuyekezi baphonononge ukuthembeka kwephepha, kwaye kulungele ulutsha olukhwankqisayo ukuphinda ulinge lakho.

Nangona iimfuniselo zebhayoloji zinzima, ziphezulu.Ukuba awulumkanga, unokulibhukuqa ihlabathi.Umzekelo, ukwenza i-SARS ibe yingxaki ye-biochemical, okanye ukwenza irayisi ye-hybrid ukugcina abantu abazibhiliyoni ezi-1.3.Lo mfanekiso ungezantsi luvavanyo lwemichiza, kufuneka uqonde ukuba uzingca kangakanani ngophando lwakho ngokujonga inkangeleko yakhe efana neDick.Ulibale, musa ukummnyama.

I-MIQE (3) - i-nucleic acid extraction.
Ukukhutshwa kwe-Nucleic acid sisiganeko esikhulu, kwaye zonke iimvavanyo zebhayoloji ye-molekyuli ziqala nge-nucleic acid extraction.Okokuqala, masikope umxholo we-MIQE kwi-nucleic acid extraction.

Ukujonga le fomu, awukwazi ukuhlala phezu komhlaba.Ifomu yimfundiso.Ukuze ube ngumfundi obalaseleyo, kufuneka ubuze ukuba kutheni.Umxholo obalulekileyo wale theyibhile ngulo: Qhubekaubunyulu, imfezeko, ukungaguquguquki, kunye nesixa sokutsalwa kwe-RNA .

Inxalenye yokuqala yeinkqubo okanye isixhobo linyathelo nucleic acid extraction.Ukuba usebenzisa i-othomathikhi ye-nucleic acid extractor ukukhupha (i-advanced, nceda uqhagamshelane nam ngokuthenga), kufuneka ubonise igama lomzekelo wesixhobo.

Igama lekhithi kunye

yeyiphi ikiti esetyenzisiweyo kwiinkcukacha zotshintsho, zeziphi ii-reagents ezikhethekileyo ezongeziweyo okanye yiyiphi imisebenzi ekhethekileyo eyenziwayo kufuneka ichazwe ngokucacileyo ukuze abanye baphinde baphinde baphinde baphinde bazame.

Abanye abantu bongeza ii-reagents ezikhethekileyo xa bekhupha iisampuli ezikhethekileyo, becinga ukuba esi sisixhobo sabo esiyimfihlo kwaye ungaxeleli abanye.Ngelixa beyigcina iyimfihlo, baphulukana nethuba lokwenza inqaku lakho liqaqambe.Musa ukuba krelekrele, kufuneka unyaniseke ngakumbi kunelizwe elidala laseZhang kuphando lwezenzululwazi, ukuba ufuna ukuba krelekrele, inqaku liya kukwenza usisidenge.

kufuneka ukhumbule inombolo yemveliso yekhithixa u-odola ikiti kwaye ubhale inqaku.Kukho amanani amabini ngokubanzi kwikiti: Inombolo yekhathalogu yekati (inombolo yemveliso, inombolo yenqaku), iLoti-inombolo yemveliso ( Isetyenziselwa ukubonisa ukuba yeyiphi ibhetshi evela kuyo).

Ukongeza, inombolo ye-CAS isetyenziswa rhoqo xa iyala i-reagents ye-biochemical, kwaye ndiya kuyenza idume kunye.Inombolo ye-CAS linani elinikwe yi-American Chemical Society kwichiza ngalinye lemichiza emitsha.Ngokuqhelekileyo, amanani amathathu aqhagamshelwe ngedash.Inombolo yeCAS kaRushui: 7732-18-5.Iikhemikhali zihlala zinee-aliases ezininzi, kodwa inombolo ye-CAS yodwa.Xa u-odola iyeza, unokujonga inombolo yayo ye-CAS kuqala.

Kufuphi nasekhaya, kutheni le nto kufuneka sizichaze ngokucacileyo ezi zinto?Ngapha koko, kukujonga umgangatho wokutsalwa kwe-RNA.Ukusetyenziswa kwezixhobo kunye neekiti kuya kwenza ukutsalwa kwe-RNA kuhambelane ngakumbi.Umlinganiselo wokutsalwa kweelabhoratri eziqhelekileyo awukho mkhulu, kwaye unokufumaneka ngeekiti.

Iinkcukacha ze-DNase okanye unyango lwe-RNase
Umba obalulekileyo we-fluorescent quantitative PCR kukuthintela ukosuleleka kwe-DNA, kwaye ungalinge ulinge ukuba kukho ungcoliseko.Ngoko ke, kunyanzelekile ukuba uchaze inkqubo oyisebenzisayo ekuqhubeni i-DNA, ukuze ubonise ukuba i-DNA kwinkqubo yovavanyo isuswe ngokupheleleyo kwaye isuswe ngokupheleleyo.imelwe ngumzobo weskim.

Umzobo we-Schematic we-RNA kunye ne-DNA
Ngokubanzi, indlela yokususa i-DNA kukunyanga i-RNA nge-DNase emva kokutsalwa.Noko ke, ezi ziindlela zakudala noko.Iikhithi zokurhweba zeRNA ziye zakwazi ukususa i-DNA ngexesha lenkqubo yokukhupha ngaphandle kokongeza i-DNase.Ngokomzekelo, uchungechunge lweekiti ezivela kwi-Foregene.

Phawula: Ukususa i-DNA ngexesha lokutsalwa kwe-RNA kuyingozi kakhulu ikrele elintlangothi-mbini, eliya kwandisa ixesha lokusebenza lokukhutshwa kwe-RNA kunye nokwandisa umngcipheko wokuthotywa kwe-RNA.Ngokusisiseko, lurhwebo phakathi kwesivuno se-RNA kunye nobunyulu.

Ukongezelela, inani le-DNase elongezelelweyo kwikholamu ye-adsorption ye-silica-based incinci kakhulu, kwaye i-DNase ephezulu kufuneka isetyenziswe ukufezekisa umphumo.I-DNase engalungiswanga ayinakutyiswa ngokukhawuleza nangokupheleleyo.Olu luvavanyo lwenqanaba lobugcisa bomthengisi.Ngokuqinisekileyo, kukho abathengisi abangaqhelekanga ngakumbi abaqhayisa ukuba i-DNA inokususwa ngaphandle kwe-DNase.Kunokuthiwa nabani na oqhayisa ukuba i-DNA inokususwa ngokupheleleyo ngaphandle kwe-DNase i-hooligan.I-DNA sisakhiwo esinemisonto ephindwe kabini, esingenakucinywa ngokuthetha nokuhleka.

Uvavanyo longcoliseko
indlela yokuvavanya: ukufunyanwa kwe-electrophoresis, i-1% ye-agarose, i-6V / cm, i-15min, ilayishwa i-1-3 ul

Nucleic acid uhlalutyo lobungakanani
idla ngokulinganiswa kusetyenziswa i-UV spectrophotometer.Makhe ndiqale ndisasaze intsingiselo yamaxabiso amathathu e-OD260, OD280, kunye ne-OD230.
·OD260nm: Yeyona wavelength yokufunxa kweyona ncopho iphakamileyo yokufunxa ye-nucleic acid, kunye nexabiso elilinganisiweyo eligqwesileyo ukusuka kwi-0.1 ukuya kwi-1.0.Ukuba akunjalo, nciphisa okanye ugxininise isampuli ukuyingenisa phakathi koluhlu.
·OD280nm: Bubude obungangelanga bokufunxa kweyona ncopho iphezulu yokufunxa yeprotheyini kunye nezinto zephenolic.
·OD230nm: Bubude obungangelanga bokufunxa kweyona ncopho iphakamileyo yokufunxa iicarbohydrates.

Okulandelayo, masithethe ngendima yesalathisi ngasinye.Kwi-A260, ingasetyenziselwa ukulinganisa isivuno se-nucleic acid.Xa i-OD260 = 1, dsDNA = 50μg / ml, ssDNA = 37μg / ml, RNA = 40μg / ml.

Ukucoceka, kufuneka sijonge imilinganiselo esiqhele ukuyibona: OD260/280 kunye ne-OD260/230.
·I-DNA ecocekileyo: I-OD260/280 iphantse ilingane no-1.8.Xa ingaphezu kwe-1.9, ibonisa ukuba kukho ungcoliseko lweRNA, kwaye xa ingaphantsi kwe-1.6, ibonisa ukuba kukho ungcoliseko lweprotheyini kunye nephenol.
·I-RNA esulungekileyo: 1.7
·OD260/230: Ingaba i-DNA okanye i-RNA, ixabiso lereferensi yi-2.5.Xa ingaphantsi kwe-2.0, ibonisa ukuba kukho ukungcoliswa kweswekile, ityuwa kunye ne-organic matter.

Ingqibelelo yeRNA

Kubaluleke kakhulu ukulinganisa ingqibelelo ye-RNA.Ngokubanzi, kuyimfuneko ukwenza ijeli ye-RNA denaturation ukujonga ukuba ukuqaqamba phakathi kwe-28S kunye ne-18S RNA bubudlelwane obuphindwe kabini.Xa ibhendi yesithathu i-5S ibonakala, ithetha ukuba i-RNA iqalile ukuthotywa, ngaphandle kwee-invertebrates.

Idatha yovavanyo lomgangatho we-RNA: Ukongeza kwezi mvavanyo zingentla, kukwakho ezinye iimvavanyo zesixhobo eziqhubela phambili ngokwemigaqo yengqibelelo ye-RNA, njengovavanyo lwengqibelelo ye-RQI ye-Experion automatic electrophoresis system, enokubona ukuba i-RNA yonakele ngokungabonakaliyo.

Kuphando lwezenzululwazi, i-fluorescent quantitative PCR luthelekiso phakathi kwejini ekujoliswe kuyo kunye nejene yereferensi yangaphakathi.Ngoko ke, kwinkqubo yokugcinwa kwesampula ye-RNA, i-RNA extraction, njl., injongo ephambili kukuqinisekisa ukuthembeka kwe-RNA.

Indlela ingqibelelo ye-RNA ichaphazela ngayo ibhalansi phakathi kwejini ekujoliswe kuyo kunye nejene yereferensi yangaphakathi inokuqondwa ngokulula ukusuka kumzobo ongezantsi.Ukuthotywa kuya kukhokelela ekungagqibekanga kofuzo, nokuba kukungaphelelanga kohlobo lwangaphakathi lwereferensi okanye ukungafezeki kohlobo olujoliswe kuyo, kuya kuba nempembelelo enkulu kwidatha.

Idayagram ecwangcisiweyo yejini ekujoliswe kuyo kunye nejini yereferensi, mayingabi yinyani

Uvavanyo lwe-inhibition (ukuba ixabiso le-CT licinezelwe phantsi koxinzelelo oluphezulu okanye oluphantsi okanye ezinye iimeko)

Ukuthatha lo mzobo njengomzekelo, amaxabiso e-Ct amagophe amahlanu alandelayo.Ukusasazwa kwamaxabiso e-CT phakathi kwee-curves azilingani, kwaye ixabiso le-Ct lilibazisekile phantsi kwee-concentrations eziphakamileyo neziphantsi, okuyiyo imeko ye-PCR inhibition.

Inqaku eliphambili: Kwinkqubo yokutsalwa kwe-RNA, kufuneka silahle iingcamango eziphosakeleyo kwaye siseke ezo zichanekileyo.

Ingcamango ephosakeleyo yile: Ukutsalwa kwe-RNA kulandela isivuno kuphela, ucinga ukuba xa umkhulu isixa se-RNA esifunyenweyo, kokukhona kungcono.Enyanisweni, xa sisenza ubungakanani, ukuba inani lemizila yemfuza alilikhulu kakhulu, asidingi RNA ingako.Ubungakanani be-RNA oyikhuphayo ingaphezulu kokwaneleyo .

Ingqikelelo echanekileyo yile:Ukutsalwa kwe-RNA kufuneka kulandele ubunyulu, ingqibelelo kunye nokungaguquguquki.Ubunyulu bunokuqinisekisa ukuba ukubhalwa kwe-reverse okulandelayo akuvinjelwanga kwaye idatha ayiyi kuchaphazeleka yiDNA.Ingqibelelo iqinisekisa ulungelelwaniso lolandelelwano ekujoliswe kulo kunye neereferensi zangaphakathi.Ukuhambelana kuqinisekisa ukulayishwa kwesampuli ezinzile.

MIQE (4) – umva ukhuphelo
Ingcamango ephosakeleyo: usukelo lomthamo wesampulu ephezulu.
Ingcamango echanekileyo: Phishekela ukuhambelana (ukuzinza), kungakhathaliseki ukuba ubungakanani be-RNA elayishiwe, ukusebenza kakuhle kokubhalwa kwe-reverse kuhlala kuhambelana, ukuqinisekisa ukuba ukungafani kwi-cDNA kunokubonisa ngokwenene ukungafani kwi-mRNA.
Sicacisa le nkqubo ngomzobo weskim:

Idayagram yeschematic yereverse transcription efficient, musa ukuba yinyani
Okokuqala, kufuneka siqonde umahluko phakathi kwenkqubo yokukhuphela umva kunye nenkqubo yePCR.I-PCR ifumana iinkqubo ezininzi zokufudumeza kunye ne-annealing, kwaye iqhekeza elijoliswe kuyo likhula ngokukhawuleza;Ngelixa ukhuphelo olubuyela umva lungenayo le nkqubo, sinokucinga ukuba ukukhutshelwa umva yinto enye ukuya-enye Ngexesha lenkqubo yophindaphindo, njengamaqhekeza amaninzi e-RNA.

njengoko kukho unokufumana amaqhekeza amaninzi eNkcukacha ye-cDNA, kufuneka iqondwe ngoku, kuba amaqhekeza amakhulu kunye amancinci aye abhalwa ngokuphindaphindiweyo, kwaye akunakwenzeka ukugxila kwisiqwenga esinye.Kwaye ngenxa yokuba isixa se-RNA sincinci ngokwentelekiso, isixa se-cDNA esifunyenweyo sikwancinci, ngokungafaniyo ne-PCR, enesiphumo sokukhulisa, ngoko ke akunakwenzeka ukubhaqa.

Iziphumo ze-cDNA electrophoresis
Okwesibini, ngokufanelekileyo, ukhuphelo olubuyela umva lwenziwa omnye ukuya komnye, kodwa akukho mbhalo ubuyela umva kuyo nayiphi na inkampani enokuphumeza esi siphumo.Ngokusisiseko, ukusebenza kakuhle kwezona zinto zibhalwayo zibuyela umva zizula phakathi kwe-30-50%.Ukuba kunjalo, singathanda ukuba ne-reverse transcription esebenzayo ezinzileyo, eyona nto sifuna ukuyibona kumzobo: ii-RNA ezi-3 zifumana ii-cDNAs ezi-2, ii-RNA ezi-6 zifumana ii-cDNA ezi-4, ngoko ke kungakhathaliseki ukuba ingakanani isampuli elayishiweyo, ukuguqulelwa kwe-reverse ukusebenza kakuhle kuzinzile.Asifuni kuyibona imeko apho ukukhutshelwa umva kokhutshelo kungazinzanga kwaye ukugxila okuphezulu kuthintelwe.

Ke, uqinisekiswa njani ukuba impumelelo yokukhuphela umva izinzile?Indlela ilula kakhulu, kufuneka wenze uvavanyo lokuthelekisa kuphela: enye kukubuyisela umva ubhalo kwi-cDNA emva kokuphinda-phindwe kabini ukudilutywa kwe-RNA, kwaye enye kukwenza ukuphinda-phinda-phinda udilution emva kokukhuphela umva kwi-cDNA, kwaye wenze i-qPCR ukubona ithambeka elifunyenweyo Ngaba liyahambelana.Njengomfundi obalaseleyo, kuya kufuneka uyiqonde ngemizuzwana.Njengoko kubonisiwe ngezantsi:

I-Dilution ye-RNA kunye ne-cDNA ukuvavanya ukuba ukusebenza kakuhle kwe-reverse transcription kuzinzile
Reverse transcriptase kunye nekhithi
Inokuba ne-PCR egqibeleleyo yobungakanani befluorescent ukuba ibe ne-reverse transcriptase egqwesileyo kunye nekhithi.I-reverse transcriptase yahlulahlulwe ngokweendidi ezimbini ngokomthombo, i-AMV okanyeI-M-MLV, kwaye ukusebenza kwabo kuyafana naleyo iboniswe kwitheyibhile.

Umsebenzi we-RNase H
I-RNase H yi-Ribonuclease H, igama lesiTshayina yi-ribonuclease H, eyi-endoribonuclease enokuthi i-hydrolyze i-RNA kwi-DNA-RNA hybrid chain.I-RNase H ayikwazi ukwenza i-hydrolyze iibhondi ze-phosphodiester kwi-DNA enye okanye i-double-stranded DNA okanye i-RNA, oko kukuthi, ayikwazi ukugaya i-DNA okanye i-RNA ephindwe kabini.Ngokuqhelekileyo isetyenziswa ekudibaneni komcu wesibini we-cDNA.

Yinto engaqhelekanga.Sithi i-reverse transcriptase inomsebenzi we-RNase H, kungekhona ukuba i-reverse transcriptase iqulethe i-RNase H, kwaye akunakwenzeka ukwahlula i-RNase H ukusuka kwi-reverse transcriptase, mhlawumbi ngenxa yokuguqulwa kwamaqela athile kwi-reverse transcriptase Lo msebenzi ubangelwa yi-reverse transcriptase.

Ke ngoko, nokuba yeyiphi na i-AMV ephezulu, umsebenzi wayo we-RNase H wehlisa isivuno se-cDNA.Ewe, abavelisi be-reagent bahlala belungiselela iimveliso zabo ukuphelisa umsebenzi we-RNase H kwi-reverse transcriptase kangangoko ukunyusa isivuno se-cDNA.
Iqondo lobushushu le-Anealing

Ubume besibini be-RNA kumaqondo obushushu ahlukeneyo
Jonga umzobo ongentla wesakhiwo sesibini se-RNA kumaqondo okushisa ahlukeneyo, kwaye usebenzise i-mFold isixhobo se-intanethi ukumisela isakhiwo sesibini seqhekeza ekujoliswe kulo phantsi kweemeko ezithile zokushisa kunye neetyuwa.Kwi-55 ° C, isakhiwo sesibini se-RNA sisenzima kakhulu, i-reverse transcriptase ayikwazi ukusebenza, kwaye isakhiwo sesibini asinakusombululwa ngokupheleleyo kude kube ngu-65 ° C, ngelixa ubushushu obuphezulu be-AMV kunye ne-M-MLV buphantsi kakhulu kunobu bushushu.
kwenziwe ntoni?Isakhiwo sesibini kukudibanisa okuhambelanayo kwetemplate ngokwayo, okukhokelela kukhuphiswano oluqinileyo phakathi kwe-primer kunye ne-reverse transcriptase kunye ne-template, okubangelwa uchungechunge lweengxaki ezifana ne-E ephantsi kunye nokuphindaphinda kakubi.

kwenziwe ntoni?Yandisa kuphela iqondo lobushushu le-anneal kangangoko kunokwenzeka .

Uninzi lwabavelisi be-reagent baphucula i-reverse transcriptase ngokusebenzisa ubunjineli bemfuza.Abanye banyusa ubushushu bokusabela, njengeJifan kunye ne-Aidelai, kwaye abanye basusa iqela elisebenzayo le-RNase H enzyme ukuphucula ubudlelwane phakathi kwe-enzyme kunye nethemplate ye-RNA.Ubudlelwane obuphezulu bunokukhuphisana ngaphandle kolwakhiwo lwesibini kwaye lufunde ngokutyibilikayo, kwaye luphucule kakhulu ukusebenza kakuhle kokukhutshelwa umva.
Inqaku eliphambili: Ukuguqulela ukuguqulwa kubaluleke kakhulu ukuqhubela phambili ukulungelelaniswa kwe-reverse transcription esebenzayo (i-enzymes kufuneka ingasebenzi kakuhle kuphela kodwa kwaye izinzile), kunokuba inani lesampuli elayishiweyo, ukuba akusiyo i-PCR enkulu ye-fluorescent quantitative quantitative, ayiyi kwenzeka konke konke.Ii-cDNA ezininzi.
Abavelisi abahlukeneyo baye benza imizamo ethile ekufuneni ukuhambelana.Umzekelo, uninzi lweenkampani ngoku zipakishe ukukhutshelwa umva njengekhithi eqhelekileyo ethengiswayo, lukhetho olulungileyo.
Umzekelo, iikhithi ze-RT Easy Series ze-Foregene:

I-RT Easy I(Master premix yekhithi yokuqala ye-cDNA yestrand)

I-MIQE (5) - ulwazi olujoliswe kwi-gene

Lo mfanekiso ungasentla uyachaza
1. Ingaba le gene iyasebenza kwiimvavanyo eziphindaphindiweyo inokuqinisekiswa ngokubanzi ngokuphindaphindiweyo.
2. Isazisi seGene, uyazi.
3. Ubude bemfuza, ubude obupheleleyo bejini ekujoliswe kulo ngokuqinisekileyo akukho ngxaki.Xa uyila ii-primers, qinisekisa ukuba ubude be-amplicon buphakathi kwe-80-200bp ukuqinisekisa ukusebenza kakuhle kokukhulisa.
4. Ulandelelwano lwengcaciso yokuthelekisa i-Blast, i-gene ekujoliswe kuyo kufuneka ifaniswe kwi-genebank ukukhusela ukukhulisa okungacaciswanga.
5. Ubukho bee-pseudogenes.I-pseudogene yi-DNA yolandelelwano efana ne-gene eqhelekileyo kodwa ilahlekelwa ngumsebenzi wayo oqhelekileyo.Ihlala ikhona kwi-multi-gene family ye-eukaryotes.Idla ngokumelwa ngu-ψ.Yikopi ye-DNA ye-genomic engasebenziyo kwi-genome efana kakhulu nolandelelwano lwe-coding gene., ngokuqhelekileyo azikhutshelwa, kwaye azinantsingiselo icacileyo ngokwasemzimbeni.
6. Isikhundla se-primers ngokumalunga ne-exons kunye nee-introns.Kwiminyaka yokuqala, xa sasombulula ingxaki yongcoliseko lwe-DNA, sasidla ngokunika ingqwalaselo kwindawo yeeprimers, exons, kunye ne-introns, kwaye ngokubanzi sithathela ingqalelo ukuyila iiprimers kuzo zonke iiintrons ukunqanda ukukhulisa iDNA.Nceda ubone umfanekiso ongezantsi: umnyama umele ii-intron, iiblues ezahlukeneyo zimele i-exons, ipinki imele iiprimers eziqhelekileyo, kunye nobomvu obuqaqambileyo bumele i-intron-spanning primers.

Uyilo, alunanyani
Sisiphi isicwangciso esigqibeleleyo esibonakala, kodwa enyanisweni, kwiimeko ezininzi, ii-primers ze-trans-intron azikho njengomlingo njengoko kucingelwa, kwaye ziya kubangela ukukhulisa okungangqalanga.Ngoko eyona ndlela yokuthintela ukosuleleka kwe-DNA kukususa iDNA ngokupheleleyo.
7. Ukuxela kwangaphambili.Ukusebenzisa lo mzekelo kwakhona, sebenzisa isixhobo se-intanethi se-mFold ukumisela isakhiwo sesibini sesiqhekeza ekujoliswe kuso kwiqondo elithile lobushushu kunye netyuwa.

Ubume besibini be-RNA kumaqondo obushushu ahlukeneyo
Isakhiwo sesibini kukudibanisa okuhambelanayo kwetemplate ngokwayo, okuya kukhokelela kukhuphiswano olunamandla phakathi kwe-primer kunye ne-template pairing, kwaye amathuba okubopha i-primer angaphantsi, okubangela uchungechunge lweengxaki ezifana ne-E ephantsi kunye nokuphindaphinda kakubi.Ngokusebenzisa uqikelelo lwesoftware, ukuba akukho ngxaki yolwakhiwo lwesibini, oko kuya kuba kuhle.Ukuba ukho, inqaku lethu elilandelayo liya kuxubusha ngokukodwa indlela yokusombulula le ngxaki.

I-MIQE (6)—qPCR Oligonucleotides

Kwi-PCR ye-fluorescent quantitative, into yokuqala oyilwa nayo yonke imihla kukukhutshwa kwe-RNA, kwaye into yesibini inokuba yi-primer design.
Okokuqala, sisajonga imigaqo malunga noyilo lwe-primer ngokoluhlu lokutshekisha lwe-MIQE.Kulula kakhulu ukuba i-scumbags inokuhleka, kwaye sinokuyigqiba kwisivakalisi esinye: fumana ukulandelelana kunye nokuma kwe-primer probe kunye nendlela yokuguqula.Kwindlela yokucoca i-primer, i-primer synthesis itshiphu kakhulu okwangoku, i-qPCR ifanele i-PAGE nangaphezulu kweendlela zokucoca, kwaye ulwazi lwesixhobo sokudibanisa alubalulekanga.Abantu abaninzi bebesenza iiprimers amashumi eminyaka kwaye abazi ukuba i-synthesizer yi-ABI3900.
Ngokumalunga nemigaqo yoyilo lwe-primer, akufuneki ukuba uyicengceleze ngentloko, kuba uninzi lwesoftware yoyilo lwe-primer okanye izixhobo ze-intanethi zinokukhathalela ezi ngxaki (isixhobo esicetyiswayo se-intanethi primer3.ut.ee/), kunye ne-99.999% yoyilo lwe-primer alwenziwanga ngesandla Khangela, umbhali ngamanye amaxesha uyila amakhulu eeprimers ngosuku, ukuba ufunda enye nenye, iya kuba yi-cross-eyed.
Jonga nje la manqaku alandelayo emva kokuba iiprimers ziyilwe:
1. I-design primers kufuphi ne-3 ekupheleni: Kwimeko yokusebenzisa i-oligo dT primers ye-cDNA yokuqala ye-strand synthesis, ngokuqwalasela ukusebenza kakuhle kwe-reverse transcription kunye ne-RNA ingqibelelo, ii-primers eziyilwe kufuneka zenziwe kufutshane ne-3 'ekupheleni ukuphucula ukusebenza kakuhle kwe-amplification.Sebenzisa umfanekiso ukuchaza ngolu hlobo lulandelayo (akukho ndlela yokuqonda oku):

Kutheni kufuneka iiprimers ziyilwe kufutshane nesiphelo se-3, akufanele kube yinyani
2. Ixabiso le-TM: Ixabiso le-Tm li-55-65 ° C (kuba umsebenzi we-exonuclease uphezulu kwi-60 ° C), kunye nomxholo we-GC kwi-40% -60%.
3. UKUQHUBEKA: Ukuze kuthintelwe ukukhuliswa okungachanekanga kwe-genome, i-Blest kufuneka isetyenziselwe uqinisekiso olongezelelweyo.

MIQE(7)—qPCR inkqubo

1. Ikhithi ye-qPCR
Ngokweemfuno ze-MIQE, kufuneka sichaze ngokucacileyo iimeko zokusabela ezipheleleyo kwinqaku, kubandakanywa ukucwangciswa kwenkqubo yokusabela kwe-PCR, yiyiphi ikiti esetyenziswayo, ngubani umenzi, indlela yokusabela enkulu kangakanani, ingaba indlela yedayi okanye indlela yokuhlola isetyenzisiweyo, izicwangciso zeprogram yePCR.Abaqhubi bamagqala baya kufumanisa ukuba okoko nje ikiti ikhethiwe, ulwazi olungentla luzimisele ngokusisiseko.
Okwangoku, ukwenziwa kunye nokuveliswa kweekhithi ze-PCR zobungakanani befluorescent bubuchwephesha obukhule kakhulu.Okoko ungakhethi abavelisi ababi kakhulu, amathuba okuba iingxaki azikho phezulu, kodwa sisafuna ukwabelana nawe ngamanqaku ambalwa:
I-enzyme ye-Taq eshushu:Eyona nxalenye ibalulekileyo ye-PCR yi-enzyme ye-Taq eshushu.Ii-enzymes ezishushu eziqalayo kwimarike zohlulwe ngokubanzi zibe ziindidi ezimbini, enye yi-enzayim eguqulwe ngokwekhemikhali eshushu yokuqalisa (unokuyicingela njengokufakela iparafini), kwaye enye yi-Enzayim eshushu yokuqalisa ukuguqulwa kwe-antibody (i-antigen-antibody ebophayo).Ukuguqulwa kweekhemikhali yindlela yokuqala yokutshisa i-enzymes.Xa ubushushu obuthile bufikelelwe, le enzyme iya kukhulula umsebenzi wayo.I-antibody-modified hot-start enzyme isebenzisa iindlela zebhayoloji ukuvala umsebenzi we-enzyme.Xa ubushushu obuthile bufikelelwe, i-antibody iya kwenziwa denatured kwaye ingasebenzi njengeprotheni, kwaye umsebenzi we-enzyme uya kudlalwa.

Nangona kunjalo, yintoni ukusetyenziswa koku?Oku kunjalo, umsebenzi wokukhutshwa kwe-antibody-modified enzymes ukhawuleza kunalowo wee-enzymes eziguqulwe ngamachiza, ngoko ke ngokobuntununtunu, i-antibody-modified enzymes ine-advanteji encinci, ukuze kungabikho ngokusisiseko ii-enzymes eziguqulwe ngamachiza kwiikhithi kwimarike.Ukuba kukho, ke iteknoloji yalo menzi isabambekile kwixesha lewaka leminyaka.
Ugxininiso lwe-Magnesium ion:I-magnesium ion concentration ibaluleke kakhulu kwi-PCR reaction.Ukugxininiswa kwe-ion ye-magnesium efanelekileyo kunokukhuthaza ukukhululwa komsebenzi we-enzyme ye-Taq.Ukuba ugxininiso luphantsi kakhulu, umsebenzi we-enzyme uya kuncitshiswa kakhulu;ukuba ugxininiso luphezulu kakhulu, i-enzyme-catalyzed non-specific amplification iya kwandiswa.Ukuxinwa kwee-ion ze-magnesium kuya kuchaphazela ukuxutywa kwee-primers, ukushisa okunyibilikayo kwethemplate kunye neemveliso ze-PCR, ngaloo ndlela kuchaphazela isivuno samaqhekeza anyusiweyo.Ukuxinwa kwee-ion ze-magnesium ngokuqhelekileyo kulawulwa kwi-25mM.Ngokuqinisekileyo, kwikiti elungileyo, ukuxinwa kwee-ion ze-magnesium kufuneka kulawulwe kakuhle.Abanye abathengisi bongeza i-agent ye-magnesium ion chelating kwi-reagent, enokuthi iphumelele umphumo wokulungelelaniswa ngokuzenzekelayo kwe-magnesium ion concentration.
Ugxininiso lwedayi yeFluorescent:Idayi ye-Fluorescent, eyi-SYBR Green esidla ngokuyisebenzisa, ikakhulu ivelisa i-fluorescence ngokuzibophelela kwi-groove encinci ye-DNA ephindwe kabini, kuba ukubophelela kwedayi kwi-DNA enemisonto ephindwe kabini ayithethi ngokuthe ngqo, oko kukuthi Logama nje i-DNA ephindwe kabini idityaniswe nayo, i-DNA idibanisa i-fluore kunye ne-fluore template yenza uphawu lwangasemva.
I-PS: Ngenxa yeempawu zayo zokukhanya, iimveliso kwimarike zipakishwa ngokubanzi kwiityhubhu ze-centrifuge ezimdaka (njengoko kubonisiwe kumfanekiso ongezantsi).Nangona kunjalo, oku kuya kuhlangabezana nengxaki.Kunzima ukubona ukuba ulwelo lufunxiwe na xa kusenziwa isampulu.Kule nkalo, i-Qingke ngokwenene isebenziseke kakhulu (njengoko kubonisiwe kumfanekiso ongezantsi), kwaye ityhubhu ecacileyo ifakwe kwi-opaque tin bag.Emva koko yifake kwibhegi ye-tin, uthathele ingqalelo ukukhululeka kokuphepha ukukhanya kunye nesampuli.Kufuneka ukhethe inombolo yemveliso eyiyo.I-TSE204 bubukho obungabizi kakhulu, nto leyo indenza ndifune ukutyala ingca.

Ukugxininiswa kwedayi ye-fluorescent nayo ibaluleke kakhulu.Ukuba i-concentration iphantsi kakhulu, i-curve yokukhulisa ayiyi kunyuka kwinqanaba elilandelayo kwaye ayifezekanga;ukuba ugxininiso luphezulu kakhulu, luya kubangela ukuphazamiseka kwengxolo.Ekubeni i-PCR ye-fluorescent quantitative ixhomekeke kakhulu kwixabiso le-CT, ukuba ukuxinwa kwedayi ye-fluorescent ayilungiswanga ngokufanelekileyo, indawo ephantsi ingcono kunomgangatho ophezulu.Ngokuqinisekileyo, i-concentration yedayi efanelekileyo iyona nto ingcono.

ROX: Iidayi ze-ROX zisetyenziselwa ukulungisa iimpazamo zesignali ye-fluorescence kakuhle.Abanye abavelisi bezixhobo bafuna ulungelelwaniso, ngelixa abanye bengafuni.Umzekelo, ukusetyenziswa kweThermo Fisher Scientific's Real Time PCR isixhobo sokukhulisa ngokuqhelekileyo kufuna ulungelelwaniso, kubandakanywa 7300, 7500, 7500Fast, StepOnePlus, njl. Imiyalelo yekhithi ngokubanzi iya kuyichaza.
I-Foregene's qPCR Mix ikwaqulethe idayi ye-ROX, elungele ukusetyenziswa kwiimodeli ezahlukeneyo.

Ixesha lokwenyani PCR Kit-Taqman

Unyango olubuthathaka lwe-hydrogen bond: Unyango lweebhondi ze-hydrogen ezibuthathaka ngumcimbi wobugcisa.Akukho nto ifunde iincwadana zeekiti ezininzi, kodwa akukho namnye kubo okhankanya esi sihloko.Enyanisweni, ibaluleke kakhulu.Ukudibaniswa kweziseko ikakhulu kuxhomekeke kumandla ebhondi ye-hydrogen.Iibhondi ze-hydrogen ezomeleleyo zikwandiswa okuqhelekileyo, kwaye iibhondi ze-hydrogen ezibuthathaka zikhokelela ekukhuliseni okungangqalanga.Ukuba iibhondi ze-hydrogen ezibuthathaka azikwazi ukupheliswa kakuhle, ukukhulisa okungaqhelekanga akunakuphetshwa.Ngaphakathi komda wombhali, ziinkampani ezimbalwa kuphela eziye zaqaphela le ngxaki.Xa uthengela ikiti, unokubhekisela ekubeni uqwalasele isisombululo kulo mbandela wekiti ofuna ukuyikhetha.

Umthamo wokuphendula: Inkqubo ye-20-50ul isetyenziswa ngokuqhelekileyo, kwaye amanani amancinci angabangela iimpazamo.Ngokubanzi, imiyalelo yekhithi iya kucebisa ukusetyenziswa kwemithamo yokusabela kwePCR.Musa ukuba krelekrele kwaye usebenzise imiqulu emincinci ukonga iindleko.injongo ye.Umthamo ocetyiswayo ngabathengisi ngokwenene uvavanyiwe, kwaye mhlawumbi abanako ukucombulula ingxaki yeempazamo ezibangelwa yimiqulu encinci.
2. Umenzi kunye nenqaku lenombolo yepleyiti yetyhubhu
Wonke umntu uyazi umgaqo we-fluorescent quantitative PCR.Ukuqokelelwa kwe-fluorescence kwenziwa ikakhulu ngee-PCR tube caps.Xa ukhetha izinto ezisetyenziswayo ze-PCR, nikela ingqalelo kumanqaku amabini: ukuhanjiswa kokukhanya okulungileyo kwaye kufanelekile kwisixhobo.Ngokuqhelekileyo, iibhodi kunye neebhubhu zeempawu eziqhelekileyo zilungile, kodwa kufuneka ukhethe ngokucophelela malunga nokulungelelanisa, ngaphandle koko awuyi kukwazi ukusebenzisa isixhobo.

4. Ulwazi lwenqanaba eliphezulu

MIQE (8)—qPCR ukuqinisekiswa
Le yeyona nto iphambili kwiqPCR!Maninzi amaqhawe awele esantini apha.Ewe kunjalo, kuyenzeka ukuba ube nethamsanqa kwaye iijini ozifundileyo zilula, ngenxa yoko udada kumqolomba womkhenkce uhamba nomoya.Ulwazi loqinisekiso lwe-qPCR lwenzelwe ukuvavanya ukuthembeka kwedatha.Sidwelisa iinkcukacha eziyimfuneko zokuqinisekisa ngolu hlobo lulandelayo:

1.Uvavanyo oluthile
Ukuchaneka kokukhulisa i-gene ekujoliswe kuyo kuvavanywa ngokujonga ukuba umfanekiso we-electrophoresis uyibhendi enye;ukuqinisekiswa kokulandelelanisa;ijika elinyibilikayo ukubona ukuba imephu encophoyo ayiyodwa;ukuqinisekiswa kwe-enzyme yokugaya kunye nezinye iindlela.
Apha, sigxininisa kwi-tyena uhlalutyo non-specific amplification usebenzisa indlela ukunyibilika curves.Ngokuqhelekileyo, xa siqulunqa ii-primers, ubukhulu beqhekeza lemveliso bufuneka ukuba bube kuluhlu lwe-80-200bp, okwenza ubushushu obunyibilikayo bemveliso ye-PCR 80-85 °C.Ke ngoko, ukuba kukho iincopho eziziintlobo ngeentlobo, kufuneka kubekho ezinye iimveliso zokukhulisa isandi ezingezizo ezodwa;ukuba incopho ibonakala ngaphantsi kwe-80 ° C, ngokuqhelekileyo ithathwa njenge-primer dimer;ukuba incopho ibonakala ngaphezu kwe-85°C, ngokuqhelekileyo ithathwa njengongcoliseko lwe-DNA okanye ukukhulisa okuNgenaso ngqo kwamaqhekeza amakhulu.
Qaphela: Ngamanye amaxesha kubakho incopho enye kuphela kuma-80°C.Ngeli xesha, le ngcamango kufuneka ithotyelwe.Kusenokwenzeka ukuba iziphumo zokwandiswa kwazo zonke ii-primer dimers.

Igophe elinyibilikayo eliqhelekileyo (incopho enye engenazo izandiso ezingazodwa)

Igophe lokunyibilika elinengxaki (ukwandiswa okungangqalanga kweencopho ezingeyonyani)
【Uhlalutyo lwemeko】

Kukho incopho ephambili, kodwa i-primer dimer inzulu
Igophe elinye lokunyibilikisa elikwincopho enye kulo mzobo ungezantsi unokukhohlisa ngokulula amehlo akho, ucinga ukuba luvavanyo olugqibeleleyo, kodwa umphumo awulunganga ngokupheleleyo.Ngeli xesha, kufuneka sijonge ubushushu obunyibilikayo.Iqondo lokushisa eliphezulu lingaphantsi kwe-80 ° C, eyi-primer-dimer ngokupheleleyo.

Akukho qhekeza ekujoliswe kulo, zonke ii-primer dimers
Apha, mntakabawo akanakuyeka.Umfanekiso ongezantsi ngumfanekiso othathwe kunye nefowuni ephathwayo ethunyelwe kum yi-scumbag.I-reagents ayisebenzisayo zonke ziimpawu ezisetyenziswa ngokuqhelekileyo kwishishini.Watshintsha ukusuka kwi-T-prefix brand ukuya kwenye i-T-prefix brand.Ndicinga ukuba sele uyiqikelele.Lo mkrweqe wadanduluka kum: “Isixhobo esisetyenziswe kumfanekiso wokuqala silunge kakhulu, yaye incopho ayiyodwa.Kamva, emva kokusebenzisa i-reagent oyicetyisileyo, iba njengomfanekiso wesibini, kunye neencopho ezixubileyo.Undiphethe kakubi.“
Yahlula iigrafu ezimbini.Ekuboneni kokuqala, enye inencopho enye, kwaye enye inencopho ephindwe kabini.Ubuvuvu, incopho enye ilungile.Ngaba yinyani leyo?
Okubi ngaphezu kweDou E, ukuba ndibeka imifanekiso emibini kumfanekiso ongezantsi, uya kuqonda ngokukhawuleza.Enyanisweni, sikhubazeka ngokulula ngolu hlobo lomfanekiso.Emva kohlalutyo olucokisekileyo, sifumene ukuba: incopho yomfanekiso wokuqala i-75 ° C, eyi-primer dimer ngokupheleleyo;incopho yomfanekiso wesibini ibonakala kwi-75 ° C kunye ne-82 ° C, ubuncinane kukho Imveliso ibonakala.

Imifanekiso yeempendulo zabafundi
Ke ingxaki esisiseko ayiyongxaki yee-reagents, kodwa yingxaki yoyilo lweprimer.Kwangaxeshanye, ikwangqina ukuba ezinye iimpawu ezinkulu azikho kumgangatho wentsimbi, kwaye ikwangqina into eyathethwa ngumntakwethu ngaphambili: Ayisiyiyo i-reagent brand exhasa inqaku lakho.Linqaku lakho elixhase uphawu lweereagents.Khawucinge nje, ukuba i-scumbag ayizange itshintshe i-reagents, idatha engafanelekanga iya kuthunyelwa kwiphephancwadi, kwaye kuya kwenzeka ntoni kuya kuba yintlekele.
2. Ixabiso le-Ct lolawulo olungenanto
Musa ukuchaza, ukuba ulawulo olungenanto lunexabiso le-Ct, akusiyo ungcoliseko?Nangona kunjalo, kusafuneka uqonde ukuba loluphi ulawulo olungenanto lunexabiso le-Ct.Ukuba yi-NTC, kuthetha ukuba kukho i-DNA yangaphandle efana nokungcoliswa kwe-reagent.Ukuba yi-NRT, ithetha ukuba i-RNA ekhutshiweyo inosulelo lwe-DNA.
3. Ijika eliqhelekileyo
Kubandakanya i-slope kunye nefomula yokubala, ukusebenza kakuhle kwe-PCR kungabalwa ngefomula.Umfuniselo ogqibeleleyo ufuna ukuthambeka kwegophe elisemgangathweni ukuya ku-3.32, kunye ne-R² ukuya ku-0.9999.
4. Uluhlu oluguquguqukayo olunomgca
Uluhlu oluguquguqukayo lwempendulo lumgca.Ngokwethemplate esetyenziselwa ukuvelisa i-curve esemgangathweni, uluhlu oluguquguqukayo kufuneka lubandakanye ubuncinane i-5 i-concentration gradients, kwaye ubeke ingqalelo ekutshintsheni kwamaxabiso e-Ct kwii-gradients eziphezulu kunye ne-low concentration gradients.
5. Ukuchaneka kokufunyanwa
Utshintsho kwiziphumo ze-qPCR, oko kukuthi, ukuphindaphinda okulambathayo, oko kukuthi, ukungachaneki kakuhle, kubangelwa zizinto ezininzi, kubandakanya ubushushu, uxinzelelo, kunye nokusebenza.Ukuchaneka kwe-qPCR kudla ngokuba lula ukulawulwa njengoko inani lekopi liyancipha.Ngokunqwenelekayo ngaphakathi-kokuzama ukwahluka, oku kwahluka kobugcisa kufuneka kwahluke kukwahluka kwebhayoloji, kwaye iziphindaphindo zebhayoloji zinokulungisa ngokuthe ngqo iyantlukwano yezibalo kwiziphumo ze-qPCR phakathi kwamaqela okanye unyango.Ngokukodwa kwiimvavanyo zokuxilonga, ukuchaneka kwe-inter-assay (ukuphindaphinda) kwiindawo zonke kunye nabaqhubi kufuneka kuxelwe.
6. Ukufumanisa ukusebenza kakuhle kunye neLOD (kwi-multiplex qPCR)
I-LOD yeyona nto iphantsi yoxinaniso lwe-95% yeesampulu ezilungileyo ezifunyenweyo.Ngamanye amazwi, ugxininiso lweLOD oluqulethwe kwiseti yethagethi yemfuza ephindaphindwayo akufunekanga ibe ngaphezu kwe-5% yeziphumo ezingaphumeleliyo.Xa uhlalutya i-multiplex qPCR, ngokukodwa ukufumanisa ngaxeshanye ukuguqulwa kwamanqaku okanye iipolymorphisms, i-multiplex qPCR idinga ukubonelela ubungqina bokuba ukuchaneka kweengcezu ezininzi ezijoliswe kuyo akuphazamisi kwi-tube enye, ukufunyanwa okuphindaphindiweyo kunye nokufunyanwa kwetyhubhu enye Ukuphumelela kunye ne-LOD kufuneka ifane.Ngokukodwa xa iijene ezijoliswe ekugxilweni okuphezulu kunye neejene ezijoliswe ekugxilweni okuphantsi zikhuliswa ngaxeshanye, le ngxaki kufuneka inikwe ingqalelo.
Iingxaki kunye nezisombululoNgokubanzi, iingxaki ezihlala zidibana kwi-qPCR debugging zijolise kule miba ilandelayo:
·ulwandiso olungangqalanga
·Ukhetho olunzima lokugxininiswa kwe-primer kunye nengxaki nge-primer-dimers
·Iqondo lobushushu lobushushu alichanekanga
·Ulwakhiwo lwesibini luchaphazela impumelelo yokwandisa
ulwandiso olungachazwanga
non-specific amplificationkwenzeka , ngokuqhelekileyo kuqwalaselwa ukuba ngaba uyilo lwe-primer alufanelekanga, kodwa ukuba awungxamanga ukutshintsha i-primers, ungazama ezi ndlela zilandelayo kuqala (umgaqo uqhotyoshelwe):
•Yandisa iqondo lobushushu le-annealing – zama ukwenza iibhondi zehydrogen ezibuthathaka zingakwazi ukuzigcina;
·Nciphisa ixesha lokwaluka kunye nokwandisa – nciphisa amathuba okuba buthathaka kweebhondi zehydrogen;
·Ukunciphisa ukugxininiswa kwe-primer - ukunciphisa ithuba lokubopha ii-primers ezingafunekiyo kunye nemimandla ekujoliswe kuyo;
Ukusebenza kokukhulisa okuphantsi
Imeko echaseneyo ne-non-specific amplification - low amplification performance , kunye nemilinganiselo yokujongana nomgangatho ophantsi wokukhulisa i-amplification ichasene nje:
Yandisa ixesha lokwandiswa kunye nexesha lokwandisa;
·Tshintshela kumanyathelo amathathu ePCR kwaye unciphise iqondo lobushushu le-anneal;
·Ukwandisa ugxininiso lweprimer;
Ps: Abafundi abaninzi abaphumeleleyo abazalwe kwiminyaka engama-90 abafuni ukufunda indlela yokulungisa iimvavanyo, kwaye banethemba lokuba ikhithi inokusombulula ngokupheleleyo ingxaki (ukuba ufuna ukuya kwinkampani ye-reagent ukwenza uphando kunye nophuhliso emva kokuphumelela), enyanisweni, abavelisi be-reagent nabo bacinga ngale ndlela, ndiyathemba ukuba isidenge Ingasetyenziswa xa uyifumene, ngoko ke abavelisi be-reagent baye bachitha umzamo omkhulu wokusombulula ingxaki yokungaphumeleli. Imiba yokufunxa ye-H-bond.Ukuyicombulula ngokulula ingxaki, izidenge kusafuneka zifunde ukuqaliswa kwenkampani ye-reagent ukubona ukuba kukho into ethatha iibhondi ze-hydrogen ezibuthathaka.
Ukhetho olunzima lokugxininiswa kwe-primer kunye neengxaki nge-primer-dimers
Indlela yoku-1: Xa sithetha ngokubanzi, imiyalelo yekhithi ye-qPCR inezixokelelwano ezicetyiswayo kunye nogxininiso lweprimer olucetyiswayo.
Indlela yesi-2: Ukulungisa ingxaki ngokuseta i-primer concentration gradient.Umfanekiso ongezantsi ubiwe kwinkampani ukubonisa.Lo mzobo ungezantsi ubonisa iziphumo zobungakanani be-fluorescence ezenziwe nge-primer concentration gradients (100nM, 250nM, 500nM) kunye ne-template ezine gradients (0.1ng, 1ng, 10ng, 100ng).Ixabiso le-Ct leziphumo zovavanyo licwangciswe ngolu hlobo lulandelayo:

I-Primer Concentration Selection Dibanisa ingxinano nganye yeprimer ibe ngumgca ngolu hlobo lulandelayo:

Ukhetho lokugxininiswa kwe-primer lucacile, ubudlelwane bomgca we-primer yoxinaniso lwe-100nM kunye ne-250nM lungcono, kwaye ubudlelwane bomgca we-primer concentration ye-500nM ihlupheke kakhulu.Kwi-100nM kunye ne-250nM, ixabiso le-Ct le-250nM lincinci, ngoko ke i-primer concentration ye-primer i-250nM.Ngokuqhelekileyo ii-primer-dimers ezinzima zingabonwa kwi-melting curve.Kuthekani ukuba i-primers eyilelwe ayikwazi ukuphepha i-primer-dimers?
Indlela yesi-3: Nciphisa inani lee-primers kunye nokwandisa ukushisa kwe-annealing (akukho mfuneko yokuchaza).
Ixabiso leempirical lobushushu be-annealing yi-60°C.Ukuba awuqinisekanga, ungakhetha njani iqondo lobushushu elilungele ngakumbi?Impendulo iyafana nokukhetha i-primer concentration -uvavanyo lwegradient.Thatha umfanekiso kwinkampani yeBio-rad ukubonisa ingxaki.Ukwandiswa kweqhekeza elithile ekujoliswe kulo, seta i-gradients ezisibhozo zobushushu, nganye iphindaphindwe kathathu, kwaye ijika lokukhulisa elifunyenweyo ngolu hlobo lulandelayo:

Ukhetho lobushushu be-annealing :
· 70 ° C, 69 ° C—Ngokusisiseko, iiprimers azikwazi ukudityaniswa, ngoko akukho nto yokukhulisa.
· 67.3 ° C - Kukho inani elincinci lokukhulisa ekuqaleni, kwaye ixabiso leCt likhulu.
·64.5°C——Ixabiso le-Ct liyancipha.
Ku-60.7 ° C, 58.0 ° C, 56.2 ° C, kunye ne-55.0 ° C, ixabiso le-Ct ngokusisiseko lithande ukuzinza, kodwa ixabiso lokugqibela le-fluorescence lahlukile.
Indlela yokukhetha?Umgaqo: Umgaqo wokuqala lixabiso eliphezulu le-Ct.Ngexabiso elifanayo le-Ct, khetha ubushushu obuphezulu bokunqanda ukunyuswa kwe-dimerization kunye nokukhulisa okungangqalanga.Nangona kukho ixabiso eliphezulu le-fluorescence kwi-55 ° C, kunokubakho i-dimers okanye i-amplification engacaciswanga kuyo.
Kodwa ukuba uhlakaniphile njengawe, ngokuqinisekileyo uya kucinga: Ukuthetha ngengqiqo, ukuba i-PCR iphendule ngokuthe ngqo, nje ukuba i-primer concentration idlula imfuno encinci, amanqaku aphezulu kunye aphantsi akufanele abe nefuthe, njengedayi ze-fluorescent kunye ne-dNTPs.Enyanisweni, nje ukuba iqondo lokushisa le-annealing liphuculwe ngokufanelekileyo, umphumo wogxininiso lwe-primer kwixabiso le-Ct ngokwemvelo liya kuncitshiswa.

Ubushushu be-annealing buphuculwe ngokufanelekileyo, kwaye umphumo we-primer concentration kwi-CT uya kuncitshiswa
Ubume besibini buchaphazela ukusebenza kakuhle kokukhulisa
Masithathe umfanekiso kwi-Bio-rad ukubonisa ingxaki.Ikwayila i-gradient yobushushu ukukhulisa i-gene enesakhiwo sesibini.

Isakhiwo sesibini siyavela
Ingabonwa ukuba njengoko i-gradient yeqondo lokushisa iyancipha, iimveliso ziqala ukubonakala kwaye ixabiso le-Ct liqhubela phambili, lifikelela kwixabiso elincinci kwi-60.7 ° C, kwaye ke njengoko iqondo lokushisa liyancipha, ixabiso le-Ct liba likhulu.Ngokwahlukileyo, njengoko iqondo lokushisa linyuka, isakhiwo sesibini sivula kunye nokusebenza kakuhle kokukhulisa.Emva kokufikelela kubushushu obuthile, ukonyusa iqondo lobushushu akunakuphucula ukusebenza kakuhle kokukhulisa.Ngenxa yokuba ii-primers azikwazi ukuhlanganiswa ngokuzinzileyo ngeli xesha.Ngoko ke,khangela ubushushu ngexabiso eliphantsi le-Ct, elona qondo lobushushu lilungileyo lokukhulisa itemplate yesakhiwo sesibini!Ngokuqinisekileyo, izidenge ezihlakaniphile kufuneka zazi ukuba ukuba akukho mfuneko, kukulungele ukutshintsha i-primers kwaye ugweme ummandla wesakhiwo sesibini.
5. Inqanaba lesicelo
I-MIQE-Uhlalutyo lwedatha

Uhlalutyo lwedatha lunikezelwa ikakhulu sisixhobo se-PCR sobungakanani befluorescent.Kwinqaku elidlulileyo, umsebenzi omningi wokuhlalutya idatha owenziweyo, njengolawulo olungenanto, oluchazwe kuyilo lovavanyo.Iijini zezalathiso zangaphakathi, amanani aphindayo, njl. njl. ziye zacaciswa., apha sichaza ikakhulu usetyenziso lwe-qPCR.
I-qPCR isetyenziswa ngokubanzi, kwaye ukuqinisekiswa kovavanyo kunye nokuxilongwa kwe-nucleic acid zezona meko zisetyenziswa kakhulu.
ubungakanani obupheleleyo
Ilogi (ingqwalasela yokuqala) inobudlelwane bomgca kunye nenani lemijikelo.Igophe elisemgangathweni linokutsalwa ukusuka kumgangatho kunye nenombolo yekopi yokuqala eyaziwayo, oko kukuthi, ubudlelwane bomgca wempendulo yokukhulisa inokufumaneka.Ngokwexabiso le-Ct yesampuli, ukugxininiswa kwisampulu ingabalwa.Isixa seethemplethi eziza kubandakanywa.

Indlela yokubala yoMyinge opheleleyo
Ubungakanani obupheleleyo kufuneka busekelwe kwigophe eliqhelekileyo.Ukwenza ijika eliqhelekileyo, umgangatho uyafuneka.Ngokuqhelekileyo, umgangatho yiplasmid efunyenwe ngokubumba i-gene ekujoliswe kuyo.Kutheni i-plasmid?Ngenxa yokuba i-DNA ye-plasmid ejikelezayo yeyona nto izinzile.Nciphisa imveliso esemgangathweni kwi-5 ukuya kwi-6 i-gradients ngokomlinganiselo ophindwe kabini (i-10-fold dilution), kwaye ubeke ingqalelo kwi-uniform xa uhlambulula.Vumela ixabiso le-Ct liwele phakathi kwe-15-30.

Ukulungiselela okusemgangathweni
Ngexesha elifanayo, isampuli ekufuneka ihlolwe nayo kufuneka ihlanjululwe ngokufanelekileyo (khumbula i-dilution factor), kunye nexabiso le-Ct kufuneka liwele phakathi kwe-15-30.Imveliso eqhelekileyo + isampuli eza kuvavanywa zibekwe kumatshini kunye.Emva kokubaleka, i-curve esemgangathweni yenziwe nge-standard substance, kwaye iisampulu eziza kuvavanywa zifakwe kwi-curve standard ukubala ukuxininisa.
Ubungakanani bentsholongwane yeHepatitis B yi-HBV quantification luphawu olupheleleyo oluqhelekileyo, olunokubala inombolo yekopi yentsholongwane kwi-1ml yegazi.
Ukubalwa kwenombolo yekopi
Isampulu yoxinaniso luza kuvavanywa (ng/ul) = OD260 × 50ug/ml × dilution factor
Isampulu yobunzima bemolekyuli = inani leziseko × 324
Inombolo yekopi yesampulu eza kuvavanywa (iikopi / ul) = ukuxinwa kwesampulu eya kuvavanywa / ubunzima bemolekyuli yesampuli × 6 × 1014

Indlela yokubala inombolo yekopi

Oku kungasentla yindlela yokubala yokumisela ubungakanani.Le yingxaki yemathematika enokusonjululwa emva kokuphumelela kwisikolo samabanga aphakamileyo samabanga aphakamileyo, kwaye iingxaki zemathematika zisonjululwa ngokubanzi ziikhompyuter.Ukuba awuqondi, ungeza ukunxibelelana.
ubungakanani obunxulumeneyo
Ubungakanani obunxulumeneyo busetyenziswa ikakhulu kuphando lwezenzululwazi.Zingaphi iintsholongwane ezikhoyo kwi-1ml yegazi, kwaye yintsholongwane ye-DNA, esi sisiganeko esicacileyo: ubungakanani begazi bunokugqitywa, kwaye i-virus ye-DNA izinzile.Nangona kunjalo, kunzima kuthi ukuthelekisa inani leekopi ezikhutshelweyo zohlobo oluthile egqabini, kuba kunzima ukumisela ubungakanani, ubunzima, kunye nokuthamba kwegqabi, ubungakanani be-RNA ekhutshiweyo kunzima ukufumanisa, kunye nokusebenza kakuhle kokukhuphela umva kunzima ukumisela, oko kukuthi, naliphi na inyathelo linokwenza idatha yovavanyo ibe neempazamo kwaye ayinakusetyenziswa.
Ke ngoko, ubungakanani obuhambelanayo kufuneka bazise into ethile:ijini yesalathiso yangaphakathi .
Ngamanye amazwi, ubungakanani obunxulumeneyo luthelekiso phakathi kwejini ekujoliswe kulo kunye nejene yereferensi yangaphakathi.Xa kuthelekiswa nezicubu ezifanayo kunye neseli efanayo, impembelelo yobungakanani besampulu, isixa sokutsalwa kwe-RNA, ukusebenza kakuhle kokukhuphela umva, kunye nokusebenza kakuhle kwePCR kuncinci.Ngenxa yobungakanani besampulu encinci, zombini iijeni zereferensi yangaphakathi kunye nejene ekujoliswe kuyo ziye zancitshiswa.Yiyo loo nto besigxininisa ukufana kunye nokuzinza ngaphambili.
Ufuzo lwangaphakathi lwereferensi luqhelekileyoufuzo lokugcina indlu(izakhi zofuzo ezigcina indlu), ezibhekiselele kudidi lwemfuza olubonakaliswa ngokuzinzileyo kuzo zonke iiseli, kwaye iimveliso zazo ziyimfuneko ukuze kugcinwe imisebenzi esisiseko yobomi beeseli.
Musa ukubhidanisa le ngcamango.Ufuzo logcino lwendlu luzimiselo zebhayoloji yokusebenza, ngelixa iijeni zesalathiso zangaphakathi zisigama sovavanyo lobugcisa.Ufuzo lokugcina indlu kufuneka lugqithise ukuqinisekiswa ngaphambi kokuba lukhethwe njengejene yereferensi yangaphakathi.
Umzekelo, sikhethe iijini ezininzi zokugcina indlu kulo mzobo ungezantsi ukuvavanya amanqanaba okuthetha kwabo kwiiseli zethishu ezahlukeneyo, kwaye safumanisa ukuba amanqanaba enkcazo ye-β-2-microglobulin ahluke kakhulu kulawo amanye amajini amathathu, ngoko ke akanakusetyenziswa njengezalathisi zangaphakathi.

Emva kokuqonda umsebenzi wokulungisa we-gene yereferensi yangaphakathi, ii-algorithms ezimbini zifumaneka ngenxa yokuqaliswa kwe-gene yereferensi yangaphakathi.
·indlela yegophe eqhelekileyo
·2 – △△Ct method (indlela yokuthelekisa ixabiso leCT)
Ukuba unomdla wokufunda iintlobo kunye nemisebenzi yemfuza, nceda uyeke uphando kwii-algorithms kwaye usebenzise iifomula ngokuthe ngqo, okanye usebenzise oomatshini ngokuthe ngqo;ukuba uyindoda ethe tye kwimathematika kunye nobunjineli, nceda uzive ukhululekile.
indlela yegophe ephindwe kabini
Ukulinganisa ijini ekujoliswe kuyo kunye nejeni yokugcina indlu yesampulu yolawulo kunye nesampuli ukuba ivavanywe kwigophe eliqhelekileyo, kwaye emva koko ubale ixabiso elihambelanayo ngokwefomula yokubala, eyinqanaba lokubonisa isihlobo.
Izinto eziluncedo: uhlalutyo olulula, ukwenziwa kovavanyo olulula
Ububi: Kwijini ngalinye, umjikelo ngamnye wovavanyo kufuneka wenze igophe eliqhelekileyo
Isicelo: Enye yezona ndlela zimbini ziqhelekileyo zisetyenziswa kwaye ziyamkelwa ubungakanani beendlela ezizalanayo kuphononongo lomgaqo wokuchazwa kofuzo.
Ifomula yile ilandelayo:

Imizekelo yile ilandelayo:

Bala isixa esihambelanayo ngokusekelwe kwisiphumo sobungakanani
2 – △△Indlela ye-CT (indlela yokuthelekisa ixabiso le-CT)

Izinto eziluncedo: Akukho mfuneko yokwenza igophe eliqhelekileyo
Ukungalungi: Kucingelwa ukuba ukusebenza kakuhle kokukhulisa kusondele kwi-100%;ukuphambuka okusemgangathweni ngu-<5%, kwaye i-curve esemgangathweni kunye nokusebenza kakuhle phakathi kwe-amplification nganye kucingelwa ukuba ihambelana;ukulungiswa kweemeko zovavanyo kunzima ngakumbi.
Isicelo: Enye yezona ndlela zimbini ziqhelekileyo zisetyenziswa kwaye ziyamkelwa ubungakanani beendlela ezizalanayo kuphononongo lomgaqo wokuchazwa kofuzo.

Ngokuqinisekileyo, ukusebenza kakuhle kwe-amplification ngokuqhelekileyo akunakwenzeka ukuba kube ngokugqibeleleyo 1. Indlela yokulungisa: Ukuba siyazi ukuba i-gene ekujoliswe kuyo kunye ne-reference gene ine-amplification efanayo, kodwa i-amplification ye-amplification ayilingani no-1, emva koko i-2△△Ct inokulungiswa njenge: (1+E ) －△△ ukulinganisa i-Ct, ngokomzekelo, i-Ct, ngokomzekelo, i-Ct, i-9 1.95－△△Ct
Ukuza kuthi ga ngoku, umxholo malunga ne-fluorescent quantitative PCR ufikelele esiphelweni.