I-RT-qPCR iphuhliswe kubuchwepheshe be-PCR obuqhelekileyo.Yongeza iikhemikhali ze-fluorescent (iidayi ze-fluorescent okanye i-fluorescent probes) kwindlela yokusabela ye-PCR yendabuko, kwaye ibone inkqubo yokudibanisa i-PCR kunye nenkqubo yokwandisa ngexesha lokwenyani ngokweendlela zabo ezahlukeneyo zokukhanya.Utshintsho lwesignali yeFluorescent phakathi lusetyenziselwa ukubala inani leenguqu zemveliso kumjikelo ngamnye wePCR.Okwangoku, ezona ndlela ziqhelekileyo yindlela yedayi ye-fluorescent kunye ne-probe method.

Indlela yedayi yeFluorescent:
Ezinye iidayi zefluorescent, ezifana neSYBR Green Ⅰ, PicoGreen, BEBO, njl. njl., azikhuphi kukhanya ngokwazo, kodwa zikhupha fluorescence emva kokubophelela kwigroove encinci ye-dsDNA.Ngoko ke, ekuqaleni kokusabela kwe-PCR, umatshini awukwazi ukubona isignali ye-fluorescent.Xa i-reaction iqhubela phambili kwi-annealing-extension (indlela yamanyathelo amabini) okanye inqanaba lokwandisa (indlela yamanyathelo amathathu), iintambo eziphindwe kabini zivulwa ngeli xesha, kunye ne-DNA entsha ye-polymerase Ngexesha le-strand synthesis, i-molecule ze-fluorescent zidibene kwi-dsDNA encinci ye-groove kwaye ikhupha i-fluorescence.Njengoko inani lemijikelo ye-PCR lisanda, iidayi ezininzi nangakumbi zidibanisa ne-dsDNA, kwaye isignali ye-fluorescent nayo iphuculwe ngokuqhubekayo.Thatha iSYBR Green Ⅰ njengomzekelo.
Indlela yokuhlola:
I-Taqman probe yeyona nto isetyenziswa kakhulu kwi-hydrolysis probe.Kukho iqela le-fluorescent kwi-5′ ekupheleni kweprobe, ngokuqhelekileyo i-FAM.Uphando ngokwalo lulandelelwano oluhambelana nejini ekujoliswe kuyo.Kukho iqela lokucima i-fluorescent kwi-3′ ekupheleni kwe-fluorophore.Ngokomgaqo we-fluorescence resonance energy transfer (Förster resonance energy transfer, FRET), xa intatheli yeqela le-fluorescent (i-molecule ye-fluorescent yomnikeli) kunye neqela lokucima i-fluorescent (i-acceptor molecule ye-fluorescent) ngelixa i-autofluorescence ibuthathaka.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.

Isazobe soku-1 Umgaqo-siseko we-qRT-PCR

Uyilo lokuqala
Imigaqo:

I-primers kufuneka yenziwe kwindawo egciniweyo yochungechunge lwe-nucleic acid kwaye ibe neenkcukacha.

Kungcono ukusebenzisa ulandelelwano lwe-cDNA, kwaye ukulandelelana kwe-mRNA kwamkelekile.Ukuba akunjalo, fumana indawo yoyilo lwe-cd yolandelelwano lwe-DNA.
Ubude bemveliso ye-fluorescent quantitative yi-80-150bp, ubude obude ngu-300bp, ubude be-primer buphakathi kweziseko ze-17-25, kwaye umehluko phakathi kwe-primers ephezulu kunye ne-downstream akufanele ibe mkhulu kakhulu.

Umxholo we-G + C uphakathi kwe-40% kunye ne-60%, kwaye i-45-55% iyona nto ingcono.
Ixabiso le-TM liphakathi kwe-58-62 degrees.
Zama ukuphepha i-primer dimers kunye ne-self-dimers, (ingabonakali ngaphezu kwe-4 izibini zeziseko ezihambelanayo ezilandelelanayo) isakhiwo se-hairpin, ukuba singenakuphepheka, yenza i-TG <4.5kJ / mol * Ukuba awukwazi ukuqinisekisa ukuba i-gDNA isusiwe ngexesha lokubhalwa kwe-reverse Ukucoceka, kukulungele ukuyila iiprimers ze-intron, i-Gc / i-Gc ingakwazi ukuphepha i-intron, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, i-Gc, okanye i-Gc Ulwakhiwo oluqhubekayo (2-3) iiprimers kunye ne-non-
ethile I-homology yolandelelwano olwandisiweyo ngokungafaniyo lukhethwa ukuba lube ngaphantsi kwe-70% okanye ine-homology yesiseko esi-8 ehambelanayo.
Uvimba weenkcukacha:
Ukukhangela kweCottonFGD ngamagama angundoqo
Uyilo lokuqala:
Uyilo lweprimer ye-IDT-qPCR

I-Fig2 IDT kwi-intanethi yesixhobo soyilo lwesixhobo sokuqala

Umboniso wephepha lesiphumo seFig3
Uyilo lweeprimer zelncRNA:
I-lncRNA:amanyathelo afanayo njenge-mRNA.
miRNA:Umgaqo wendlela ye-stem-loop: Ekubeni zonke ii-miRNA zilandelelwano ezimfutshane malunga ne-23 nt, ukufunyanwa kwe-PCR ngokuthe ngqo akunakwenziwa, ngoko ke isixhobo sokulandelelana kwe-stem-loop sisetyenziswa.Ukulandelelana kwe-stem-loop yi-DNA enemisonto enye emalunga ne-50 nt, enokwenza isakhiwo se-hairpin ngokwayo.I-3 'Isiphelo sinokuyilwa njengolandelelwano oluhambelana neqhekeza le-miRNA inxalenye, emva koko i-miRNA ekujoliswe kuyo inokudityaniswa kulandelelwano lwe-stem-loop ngexesha lokukhuphela umva, kwaye ubude bubonke bunokufikelela kwi-70bp, ehambelana nobude bemveliso eyandisiweyo egqitywe yi-qPCR.Uyilo lwe-primer ye-miRNA .
Ukufunyaniswa kokukhulisa ngokuthe ngqo:
Isiseko sedatha ye-intanethi: I-CottonFGD iqhuma ngokulandelelana ukufana
Ukuqhuma kwendawo: Jonga ukusebenzisa iBlast + ukwenza ukuqhuma kwendawo, i-linux kunye ne-macos inokuseka ngokuthe ngqo isiseko sedatha yendawo, inkqubo ye-win10 inokwenziwa emva kokufaka ubuntu bash.Yenza isiseko sedatha yendawo kunye nokuqhushumba kwendawo;vula ubuntu bash kwi win10 .
Qaphela: Umqhaphu we-Upland kunye nomqhaphu wesiqithi saselwandle zizityalo ze-tetraploid, ngoko ke isiphumo sokuqhushumba siya kuba yimidlalo emibini okanye ngaphezulu.Kwixesha elidlulileyo, ukusebenzisa ii-cd ze-NAU njengendawo yogcino-lwazi ukwenza uqhushululu kusenokwenzeka ukuba kufumaneke iijini ezimbini ze-homologous ezinomahluko nje we-SNP ezimbalwa.Ngokuqhelekileyo, i-genes ezimbini ze-homologous azikwazi ukwahlula ngoyilo lwe-primer, ngoko ziphathwa ngokufanayo.Ukuba kukho i-indel ecacileyo, i-primer idla ngokuyilwa kwi-indel, kodwa oku kunokukhokelela kwisakhiwo sesibini se-primer I-energy yamahhala iba phezulu, ekhokelela ekunciphiseni kokusebenza kakuhle kokukhulisa, kodwa oku akunakuphepheka.

Ukufunyanwa kolwakhiwo lweprimer yesibini:
Amanyathelo:vula i-oligo 7 → ulandelelwano lwetemplate yegalelo → vala i-sub-window → gcina → fumana iprimer kwitemplate, cinezela ctrl+D ukuseta ubude beprimer → hlalutya izakhiwo eziziisekondari ezahlukeneyo, ezifana ne-self-dimerization body, heterodimer, hairpin, mismatch, njl.Isiphumo se-primer yangaphambili silungile, akukho sakhiwo esicacileyo se-dimer kunye ne-hairpin, akukho ziseko ezihambelanayo eziqhubekayo, kwaye ixabiso elipheleleyo lamandla amahhala lingaphantsi kwe-4.5, ngelixa i-primer yangasemva ibonisa ngokuqhubekayo Iziseko ezi-6 ziyancedisana, kwaye amandla akhululekile yi-8.8;Ukongeza, i-dimer enzulu ngakumbi ibonakala ekupheleni kwe-3, kwaye i-dimer ye-4 yeziseko ezilandelelanayo ibonakala.Nangona amandla asimahla engekho phezulu, i-3′ dimer Chl inokuchaphazela ngokunzulu ukucaciswa kokukhulisa kunye nokusebenza kakuhle kokukhulisa.Ukongezelela, kuyimfuneko ukukhangela i-hairpins, i-heterodimers, kunye nokungahambi kakuhle.

Fig3 oligo7 iziphumo zokubona
Ukufunyaniswa kobuchule bokukhulisa:
Ubuchule bokukhulisa indlela yokusabela kwe-PCR buchaphazela kakhulu iziphumo ze-PCR.Kwakhona kwi-qRT-PCR, ukusebenza kakuhle kokukhulisa kubaluleke kakhulu kwiziphumo zobungakanani.Susa ezinye izinto, oomatshini kunye neeprothokholi kwi-reaction buffer.Umgangatho we-primers nawo unempembelelo enkulu ekusebenzeni kokukhulisa i-qRT-PCR.Ukuze kuqinisekiswe ukuchaneka kweziphumo, zombini i-fluorescence quantification kunye ne-absolute fluorescence quantification kufuneka ibone ukusebenza kakuhle kwe-amplification ye-primers.Kuyaqondwa ukuba Ubuchule bokukhulisa i-qRT-PCR buphakathi kwe-85% kunye ne-115%.Kukho iindlela ezimbini:
1. Indlela yegophe eqhelekileyo:
a.Xuba i-cDNA
b.Ukunyuswa kwegradient
c.qPCR
d.Umgca wokuhlehla equation ukubala impumelelo yokwandisa
2. I-LinRegPCR
I-LinRegPCR yinkqubo yohlalutyo lwedatha ye-RT-PCR yexesha langempela, ebizwa ngokuba yi-quantitative PCR (qPCR) idatha esekelwe kwi-SYBR Green okanye i-chemistry efanayo.Inkqubo isebenzisa idatha echanekileyo engekho siseko, yenza ukulungiswa kwesiseko kwisampuli nganye ngokwahlukileyo, imisela i-window-linearity kwaye isebenzisa uhlalutyo lokuhlengahlengiswa komgca ukuze ulungele umgca ochanekileyo ngokusebenzisa isethi yedatha ye-PCR.Ukusuka kwithambeka lalo mgca ukusebenza kwe-PCR kwisampula nganye nganye kubalwa.I-PCR esebenzayo kwi-amplicon nganye kunye nexabiso le-Ct ngesampulu nganye isetyenziselwa ukubala i-concentration yokuqala ngesampulu, echazwe kwiiyunithi ze-fluorescence ezingenasizathu.Ukufakwa kwedatha kunye nemveliso kusetyenziswa i-Excel spreadsheet.Isampuli kuphela
ukuxuba kuyadingeka, akukho gradient
amanyathelo ayafuneka:(Thatha i-Bole CFX96 njengomzekelo, hayi uMtshini one-ABI ecacileyo)
umfuniselo:luvavanyo oluqhelekileyo lwe-qPCR.
imveliso yedatha ye-qPCR:I-LinRegPCR inokubona iindlela ezimbini zeefayile zemveliso: i-RDML okanye i-quantification Amplification result.Enyanisweni, lixesha langempela lokubona ixabiso lenombolo yomjikelezo kunye nesignali ye-fluorescence ngumatshini, kwaye ukukhulisa kufunyenwe ngokuhlalutya ixabiso lokutshintsha kwe-fluorescence yecandelo lomgca osebenzayo.
Ukukhetha idatha: Kwithiyori, ixabiso le-RDML kufuneka lisebenziseke.Kuqikelelwa ukuba ingxaki yekhompyuter yam kukuba isoftware ayinakuqaphela i-RDML, ngoko ke ndinexabiso lemveliso egqwesileyo njengedatha yoqobo.Kucetyiswa ukuba kwenziwe uvavanyo olubi lwedatha kuqala, njengokungaphumeleli kokongeza iisampulu, njl. njl

Fig5 qPCR data ngaphandle

Fig6 ukhetho lweesampulu zomgqatswa

Ungeniso lwedatha:Vula iziphumo zolwandiso lwesiqinisekiso.xls, → vula iLinRegPCR → ifayile → funda kwi-excel → khetha iiparamitha njengoko kubonisiwe kuMfanekiso 7 → Kulungile → cofa misela iziseko

Fig7 amanyathelo okufakwa kwedatha ye-linRegPCR

Isiphumo:Ukuba akukho phindaphindo, akukho qela lifunekayo.Ukuba kukho uphinda-phindo, iqela lingahlelwa kwiqela lesampulu, kwaye igama lejini lifakwe kwisazisi, kwaye ke kwa uhlobo olufanayo lofuzo luya kufakwa ngokwamaqela ngokuzenzekelayo.Ekugqibeleni, cofa kwifayile, thumela ngaphandle, kwaye ujonge iziphumo.Ubuchule bokukhulisa kunye neziphumo ze-R2 zequla ngalinye ziya kuboniswa.Okwesibini, ukuba uyahlula ngokwamaqela, umndilili olungisiweyo wokukhulisa umlinganiselo uya kuboniswa.Qinisekisa ukuba ulwandiso lolwandiso lweprimer nganye luphakathi kwama-85% kunye ne-115%.Ukuba inkulu kakhulu okanye incinci kakhulu, oko kuthetha ukuba ukusebenza kakuhle kokukhulisa i-primer kubi.

Fig 8 Iziphumo kunye neziphumo zedatha

Inkqubo yovavanyo:
Iimfuno zomgangatho we-RNA:
Ubunyulu:1.72.0 ibonisa ukuba kusenokubakho i-isothiocyanate eshiyekileyo.I-nucleic acid ecocekileyo i-A260 / A230 kufuneka ibe malunga ne-2 .Ukuba kukho i-absorption enamandla kwi-230 nm, ibonisa ukuba kukho izinto eziphilayo ezifana ne-phenate ions.Ukongezelela, inokubonwa nge-1.5% ye-agarose gel electrophoresis.Khomba umakisha, kuba i-ssRNA ayinayo i-denaturation kwaye i-logarithm ye-molecular weight ayinalo ubudlelwane bomgca, kwaye ubunzima be-molecular abunakubonakaliswa ngokuchanekileyo.Ugxininiso: Ngokwethiyorihayingaphantsi kwe-100ng / ul, ukuba ugxininiso luphantsi kakhulu, ukucoceka ngokuqhelekileyo kuphantsi kungekhona ubude

Umfanekiso we-9 RNA ijeli

Ukongeza, ukuba isampuli ixabisekile kwaye i-RNA igxininisekile, kuyacetyiswa ukuba i-aliquote emva kokutsalwa, kwaye ihlambulule i-RNA kwi-concentration yokugqibela ye-100-300ng / ul yokuguqulwa kwe-reverse.Kwiinkqubo yokukhuphela umva, xa i-mRNA ibhaliwe, iiprimers ze-oligo (dt) ezinokubophelela ngokukodwa kwimisila ye-polyA zisetyenziselwa ukukhuphela umva, ngelixa i-lncRNA kunye ne-circRNA zisebenzisa i-random hexamer (i-Random 6 mer) i-primers ye-reverse transcription ye-RNA iyonke Kwi-miRNA, i-miRNA-specific transcription are primes neck-loop.Iinkampani ezininzi ngoku sele ziphehlelele iikhithi ezikhethekileyo.Kwindlela ye-stem-loop, indlela yomsila ilungele ngakumbi, i-high-throughput, kunye ne-reagent-saving, kodwa Isiphumo sokuhlukanisa i-miRNA yentsapho efanayo akufanele ibe yinto enhle njengendlela ye-stem-loop.Ikhithi nganye yokukhuphela umva ineemfuno zoxinaniso lweeprimers zegene-specific (stem-loops).Isalathiso sangaphakathi esisetyenziselwa i-miRNA yi-U6.Kwinkqubo ye-stem-loop inversion, ityhubhu ye-U6 kufuneka iguqulwe ngokwahlukileyo, kwaye i-front and back primers ye-U6 kufuneka yongezwe ngokuthe ngqo.Zombini i-circRNA kunye ne-lncRNA zinokusebenzisa ii-HKG njengereferensi yangaphakathi.Kwiukufunyanwa kwe-cDNA,
ukuba akukho ngxaki nge-RNA, i-cDNA nayo kufuneka ilungile.Nangona kunjalo, ukuba ukugqibelela kovavanyo kulandelwa, kungcono ukusebenzisa ijene yereferensi yangaphakathi (Reference gene, RG) enokwahlula i-gDNA kwiiCD.Ngokubanzi, i-RG yimfuza yokugcina indlu., HKG) njengoko kuboniswe kuMfanekiso 10;Ngelo xesha, ndandisenza iprotein yokugcina iimbotyi zesoya, kwaye ndisebenzisa i-actin7 equlethe ii-intron njengesalathiso sangaphakathi.Ubungakanani beqhekeza elikhulisiwe lale primer kwi-gDNA yayingu-452bp, kwaye ukuba i-cDNA isetyenziswe njenge template, yayingu-142bp.Emva koko iziphumo zovavanyo zafumanisa ukuba iNxalenye ye-cDNA ngokwenene yayingcolisekile yi-gDNA, kwaye yangqina kwakhona ukuba akukho ngxaki ngesiphumo sokukhutshelwa umva, kwaye ingasetyenziswa njenge template ye-PCR.Akunamsebenzi ukuqhuba i-agarose gel electrophoresis ngokuthe ngqo kunye ne-cDNA, kwaye i-diffuse band, engakholisiyo.

Umfanekiso we-10 cDNA ukufunyanwa

Ukumiselwa kweemeko ze-qPCRayiyongxaki ngokubanzi ngokweprothokholi yekhithi, ikakhulu kwinyathelo lexabiso le-tm.Ukuba ezinye ii-primers azenziwanga kakuhle ngexesha loyilo lwe-primer, okubangela ulwahlulo olukhulu phakathi kwexabiso le-tm kunye ne-theory 60 ° C, kucetyiswa ukuba i-cDNA Emva kokuba iisampuli zixutywe, sebenzisa i-PCR ye-gradient kunye ne-primers, kwaye uzame ukuphepha ukubeka ubushushu ngaphandle kweebhendi njengexabiso le-TM.

Uhlalutyo lwedatha

Indlela yesiqhelo ye-fluorescence quantitative PCR processing ngokusisiseko ngokwe-2-ΔΔCT.Ithemplethi yokucwangcisa idatha.

Iimveliso ezinxulumeneyo:

Ixesha lokwenyani PCR Easy^TM – Taqman

Ixesha lokwenyani PCR Easy^TM –SYBR GREEN I

I-RT Easy I (I-Master Premix ye-strand yokuqala ye-cDNA synthesis)

I-RT Easy II(I-Master Premix ye-strand yokuqala ye-cDNA synthesis ye-qPCR)