Ukuchaneka kokufunyanwa
Kwiimeko ezininzi, injongo yoyilo lwe-primer kukwandisa ubukhulu be-PCR.Oku kumiselwa yimpembelelo engaphezulu okanye engaphantsi eqikelelwayo yeenguqu ezininzi.Enye inguqu ebalulekileyo lulandelelwano kwi-3′ ekupheleni kwe-primer.
Okubalulekileyo, iimvavanyo ze-PCR ezenzelwe ukuchaneka zinokugcina ukusebenza okuphezulu kuluhlu olubanzi oluguquguqukayo, kuba uvavanyo aluvelisi mveliso yokukhulisa i-non-specific, ngaloo ndlela ikhuphisana ne-PCR reagents okanye ithintele impendulo ephambili yokukhulisa.
Ngokuqinisekileyo, kwezinye iimeko, ukuchaneka akuyona into ebaluleke kakhulu, umzekelo, xa injongo kukulinganisa ngokusondeleyo kodwa i-pathogens ehlukeneyo, ukuyila okukhethekileyo, ukulungiswa kunye nemigangatho yokuqinisekisa iyafuneka.
I-melting curve yindlela esemgangathweni yokuvavanya ukuchaneka kwee-amplicons, ubuncinci malunga nokuba ukhulise injongo enye.Nangona kunjalo, kufuneka kugxininiswe ukuba i-melting curves inokudukisa kuba, umzekelo, inokuchaphazeleka kwimiphumo edibeneyo ye-primers suboptimal primers kunye ne-low template concentrations.
P5 |Ijika elinyibilikayo libonisa utshintsho lwe-Tm olufunyenwe kwizinto ezimbini ezifunyenweyo kwiimali ezahlukeneyo zee-DNA ezijoliswe kuzo.
A. Kugxininiso oluphezulu (ad)), akukho nto icacileyo ye-primer dimer emva kokuba umlinganiselo we-qPCR ugqityiwe.Njengoko i-template concentration iyancipha ukuya kwiikopi ezingama-50 (e), imveliso engeyodwa iqala ukuvela kwaye ibe yimveliso kuphela kwezona ndawo zisezantsi (f).
B. Uvavanyo lurekhode ii-Tms ezifanayo kuzo zonke iinkalo ekujoliswe kuzo, kwaye akuzange kubekho i-primer dimer icacileyo nakwelona qondo lisezantsi (iikopi ezi-5).Xa usebenzisa ezi ndlela zimbini zokubhaqa, akukho mveliso zokukhulisa zichongiwe kwii-NTC.
I-P5 ibonisa ii-curves zokuchithwa ezifunyenwe kunye neesampuli apho i-template ikhona kwiindawo ezahlukeneyo.I-P 5a ibonisa ukuba kwiindawo ezimbini ezisezantsi, i-Tms yeemveliso ezingezona ezikhethekileyo zokukhulisa imveliso eziveliswayo zingaphantsi kunezo ze-amplicons ezithile.
Ngokucacileyo, le ndlela yokubona ayinakusetyenziswa ngokuthembekileyo ukufumanisa iithagethi ezikhoyo kwiindawo eziphantsi.
Okubangel 'umdla kukuba, ii-NTCs, oko kukuthi, iisampuli ezingenayo i-DNA kwaphela, azizange zirekhode (ezingangqalanga) iimveliso zokukhulisa ulwazi, ezibonisa ukuba imvelaphi ye-genomic DNA inokuthatha inxaxheba ekukhuliseni/kwipolymerization engangqalanga.
Ngamanye amaxesha i-primers enjalo yangasemva kunye nokukhulisa okungangcaciswanga akukwazi ukulungiswa, kodwa kaninzi kuyenzeka ukuyila indlela yokubona engenalo ukukhulisa okungangqalanga kuyo nayiphi na i-template concentration kunye ne-NTC (P 5b).
Apha, nokuba urekhoda ukunyuswa kogxininiso olujoliswe kuyo kunye neCq ye-35 kuya kuvelisa i-curve ethile yokuchithwa.Ngokufanayo, ii-NTCs azikhange zibonise zimpawu zokwandisa okungangqalanga.Ngamanye amaxesha, indlela yokuziphatha yokufumanisa inokuxhomekeka kutywala bukamama, kwaye kuphela ulwandiso olungangxengwanga luchongiwe kwizinto ezithile ze-buffer, ezinokunxulumana nokugxilwa kweMg2+ eyahlukileyo.
Ukuzinza kokufumanisa
Ukwenziwa ngcono kwe-Ta linyathelo eliluncedo kuqinisekiso lwamandla kunye nenkqubo yokuphucula ubhaqo lwe-qPCR.Inika isibonakaliso esithe ngqo sokuqina kwe-primer iseti ngokubonisa iqondo lokushisa (okanye iqondo lokushisa) elivelisa i-Cq ephantsi ngaphandle kokukhulisa i-NTC.
Umahluko ophindwe kabini ukuya kwezine kubuntununtunu ungabalulekanga kubantu abanenkcazo ephezulu ye-mRNA, kodwa kwiimvavanyo zokuxilonga, kunokuthetha umahluko phakathi kweziphumo ezingalunganga nezingalunganga.
Iimpawu ze-Ta zeeprimer ze-qPCR zinokwahluka kakhulu.Ezinye iimvavanyo azinamandla kakhulu, kwaye ukuba azenziwanga phantsi kwexabiso elichanekileyo leTa yeeprimers, ziya kukhawuleza ziwa.
Oku kubalulekile kuba olu hlobo lokufumanisa luhlala luyingxaki kwihlabathi lenene, kwaye ukucoceka kwesampulu, ukuxinwa kwe-DNA, okanye ubukho benye i-DNA busenokungabi yinto efanelekileyo.
Ukongezelela, inombolo yekopi ejoliswe kuyo inokwahluka kuluhlu olubanzi, kwaye i-reagents, izitya zeplastiki, okanye izixhobo zinokuthi zihluke kwizinto ezisetyenziselwa ukuseta uvavanyo.
P6|Iqondo lobushushu libonisa ukomelela okwahlukileyo kokubhaqwa kwe-PCR.
A. Sebenzisa i-Bioline's Sensifast SYBR mastermix (inombolo yekhathalogu ye-BIO-98050) ukwenza i-PCR kwi-cDNA elungiswe kwi-RNA yobuchopho bomntu.
B. Sebenzisa isixhobo se-Bio-Rad's CFX qPCR ukurekhoda imephu yokukhulisa kunye nonyiso lwegophe le-apalene (NM_033207, F: GCCATGGAGGAAAGTGACAGACC, R: CTCATGTGTGGGTGATCTCCTAGG).
C. Igrafu yokukhulisa kunye ne-melting curve ye-ACSBG1 (NM_015162.4, F: CTACACTTCCGGCACCACTGG, R: GTCCACGTGATATTGTCTTGACTCAG).
D. Igrafu yokukhulisa kunye nejika lokuchithwa kwe-GFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGGATCTTC).
I-E. Cqs irekhodwe kumaqondo okushisa ahlukeneyo, ebonisa umahluko kwi-Cq erekhodiweyo phantsi kwe-7C yeqondo lokushisa.
I-P 6 ibonisa umphumo oqhelekileyo wovavanyo olungathandekiyo, apho i-qPCR yenziwa kusetyenziswa i-gradient Tas phakathi kwe-59C kunye ne-67C (P 6a), isebenzisa ii-primers kwiijene ezintathu zobuchopho bomntu.
Inokubonwa kwigrafu yokukhulisa ukuba iiprimer ze-Opalin zikude nelungileyo ngenxa yokuba uluhlu lwazo lwe-Ta lumxinwa kakhulu (Umfanekiso 6b), oko kukuthi, ii-Cqs zisasazwe ngokubanzi, okukhokelela ekubeni ii-Cqs zithelekiswe kakhulu nezona Cqs zazo ziPhantsi.
Le ndlela yokubona ayizinzanga kwaye ingakhokelela kulwandiso olungenalona lufanelekileyo.Ngoko ke, ezi zibini zee-primers kufuneka zenziwe ngokutsha.Ukongezelela, uhlalutyo lwe-melting curve (i-inset) lubonisa ukuba ukucaciswa kwale ndlela yokufumanisa kunokuba yingxaki, kuba i-melting curve ye-Ta nganye iyahluka.
Indlela yokufumanisa i-ACSBG1 eboniswe kwi-P 6c yomelele ngakumbi kunendlela yokufumanisa i-Opalin apha ngasentla, kodwa isekude neyona nto ilungileyo, kwaye kunokwenzeka ukuba inokuphuculwa.
Nangona kunjalo, sigxininisa ukuba akukho nxu lumano oluyimfuneko phakathi kokuqina kunye nokuchaneka, kuba i-curve yokuchithwa eveliswa yile ndlela yokufumanisa ibonisa ixabiso eliphakamileyo elifanayo kuyo yonke i-Tas (i-inset).
Ngakolunye uhlangothi, uvavanyo oluqinileyo lunokunyamezela kakhulu, luvelisa ii-Cq ezifanayo kuluhlu olubanzi lwe-Tas, njengoko kuvavanyo lwe-GFAP oluboniswe kwi-P 6d.
Umahluko kwi-Cqs efunyenwe kwinqanaba elifanayo le-8 degrees Celsius lingaphantsi kwe-1, kwaye i-curve ye-dissolution (inset) iqinisekisa iimpawu zokufumanisa kule nqanaba lokushisa.Kuyafaneleka ukuba uqaphele ukuba i-Tas ebalwayo kunye noluhlu lwangempela lwe-Ta lunokwahluka kakhulu.
Kukho izikhokelo ezininzi ezenzelwe ukunceda abaphandi baqulunqe ii-primers ezisebenzayo, ezininzi zazo zisekelwe kwimithetho esele imiselwe kwaye ingqwalasela eninzi ihlawulwe kwi-3′end ye-primers.Ngokuqhelekileyo kunconywa ukubandakanya i-G okanye i-C ekupheleni kwe-3 kunye neziseko ezimbini ze-G okanye i-C (i-GC i-clamp), kodwa akukho ngaphezu kwezibini ze-5 zokugqibela.
Enyanisweni, le mithetho inokukhokela abaphandi, kodwa ayichanekanga phantsi kwazo zonke iimeko.
P7 |I-3'end ye-primer inempembelelo encinci kwizinto ezithile okanye ukusebenza kakuhle.
A. Isikhundla se-primers ye-HIF-1α yomntu (NM_181054.2) gene.
B. Sebenzisa i-Agilent Brilliant III SYBR utywala obungumama obuluhlaza (Cat. No. 600882) ukukhulisa izinto ezintandathu zovavanyo.
C. Igrafu yokukhulisa kunye nokunyibilika kwegophe ebhalwe yi-CFX qPCR isixhobo se-Bio-Rad kunye ne-3′end primers.Ii-NTC ziboniswa ngombala obomvu.
D. Cqs irekhodi yento nganye yovavanyo
Ngokomzekelo, umphumo kwi-P 7 uphikisana nomgaqo we-3′end.Lonke uyilo luvelisa iziphumo ezifanayo, kunye neendibaniselwano ezimbini kuphela ezikhokelela kulwandiso olungangqalanga kwi-NTC.
Nangona kunjalo, asikwazi ukuxhasa umphumo we-GC clip, kuba kule meko, ukusebenzisa i-A okanye i-T njengobuninzi beziseko ze-30 akunciphisi ukucaciswa.
Uvavanyo C, apho i-F primer iphela kwi-GGCC, yenza irekhodi ye-Cqs kwi-NTCs, ebonisa ukuba umntu unokufuna ukuphepha oku kulandelelana kwi-30-ekupheleni.Siyagxininisa ukuba ekuphela kwendlela yokumisela eyona 3′ yolandelelwano lweprimer kukuvavanya ezinye iiprimers zomvavanyo.
Ukwandiswa kobuchule
Okubalulekileyo, nangona ukuchongwa kwe-PCR engacacanga akunakuze kucace, ukusebenza kakuhle kokukhulisa kunokulungiswa kwaye kwandiswe ngeendlela ezininzi ezahlukeneyo ngokuguqula i-enzyme, utywala bukamama, izongezo, kunye neemeko zokuhamba ngebhayisikile.
Ukuvavanya ukusebenza kakuhle kokufunyanwa kwe-PCR, kungcono ukusebenzisa i-serial dilution ye-10 okanye i-5 amaxesha ekujoliswe kuyo i-nucleic acid, oko kukuthi, "indlela ye-curve standard".
Ukuba ii-amplicons ze-PCR okanye iithagethi ze-DNA zokwenziwa zisetyenziselwa ukuvelisa i-curve eqhelekileyo, ukuhlanjululwa kwe-serial kwezi thagethi kufuneka kuxutywe kunye nenani eliqhubekayo le-DNA yangasemva (njenge-DNA ye-genomic).
P8 |Ijika le-Dilution ukuvavanya ukusebenza kakuhle kwe-PCR.
A. Sebenzisa iiprimers ze-HIF-1: F: AAGAACTTTTAGGCCGCTCA kunye ne-R: TGTCCTGTGGTGACTTGTCC kunye ne-Agilent's Brilliant III SYBR I-mastermix eluhlaza (inombolo yekhathalogu 600882) ye-PCR kunye neemeko ze-curve ezinyibilikayo.
B. I-100 ng RNA yabhalwa ngokuphindaphindiweyo, ihlanjululwe ngamaxesha e-2, kwaye iisampulu ze-cDNA ezihlanjululweyo zahlanjululwa ngamaxesha angama-5 kwi-1 ng ye-DNA ye-genomic yabantu.Igophe lokunyibilika libonisiwe kwi-inset.
C. Ukusabela kwe-RT, i-dilution, kunye ne-serial dilution yaphinda yaphindwa kwisampuli yesibini ye-cDNA, kwaye iziphumo zifana.
I-P 8 ibonisa ama-curves amabini aqhelekileyo, usebenzisa indlela efanayo yokufumanisa kwiisampuli ezimbini ze-cDNA ezahlukeneyo, umphumo ufana nokusebenza kakuhle, malunga ne-100%, kunye nexabiso le-R2 likwafana, oko kukuthi, iqondo lokulinganisa phakathi kwedatha yovavanyo kunye nomgca wokubuyisela okanye i-Degree ye-linearity.
Iigophe ezimbini ezisemgangathweni ziyathelekiseka, kodwa azifani ncam.Ukuba injongo kukulinganisa ubungakanani bethagethi ngokuchanekileyo, makuqatshelwe ukuba akwamkelekanga ukunika ikopi inombolo yokubala ngaphandle kokuchaza ukungaqiniseki.
P9 |Ukungaqiniseki komlinganiselo ohambelana nobungakanani usebenzisa ijika eliqhelekileyo.
A. Sebenzisa iiprimers ze-GAPDH (NM_002046) ukwenza i-PCR kunye neemeko zokunyibilika kwegophe.F: ACAGTTGCCATGTAGACC kunye ne-R: TAACTGGTTGAGCACAGG kunye ne-Bioline's Sensifast SYBR mastermix (inombolo yekhathalogu ye-BIO-98050).
B. Itshati yokukhulisa, igophe elinyibilikayo kunye negophe elisemgangathweni elirekhodwe ngesixhobo se-Bio-Rad's CFX qPCR.
C. Igrafu ye-curve esemgangathweni kunye ne-95% yexesha lokuzithemba (CI).
D. Inombolo yekopi kunye ne-95% yexesha lokuzithemba kwamaxabiso amathathu e-Cq athathwe kwi-curve dilution.
I-P 9 ibonisa ukuba uvavanyo oluphuculweyo, ukuguquguquka kwemvelo kwi-curve eyodwa esemgangathweni malunga namaxesha e-2 (i-95% yexesha lokuzithemba, ubuncinci ukuya kwi-maximum), enokuthi ibe yintlukwano encinci enokulindelwa.
Imveliso eyeleleneyo:
Ixesha lokuposa: Sep-30-2021
