Izinto zokuqala: i-RNA
I-Quantitative reverse transcription PCR (RT-qPCR) indlela yokuhlola esetshenziswa ekuhloleni kwe-PCR kusetshenziswa i-RNA njengento yokuqala.Kule ndlela, i-RNA ephelele noma i-RNA yesithunywa (mRNA) ibhalwa kuqala ibe yi-DNA ehambisanayo (cDNA) nge-reverse transcriptase.Kamuva, ukusabela kwe-qPCR kwenziwa kusetshenziswa i-cDNA njengesifanekiso.I-RT-qPCR isetshenziswe ezinhlelweni ezihlukahlukene zebhayoloji yamangqamuzana, okuhlanganisa ukuhlaziywa kwezakhi zofuzo, ukuqinisekiswa kokuphazanyiswa kwe-RNA, ukuqinisekiswa kwe-microarray, ukutholwa kwe-pathogen, ukuhlolwa kofuzo, nocwaningo lwezifo.
Isinyathelo esisodwa kanye nezinyathelo ezimbili ze-RT-qPCR
I-RT-qPCR ingafezwa ngesinyathelo esisodwa noma ezimbili.Isinyathelo esisodwa se-RT-qPCR sihlanganisa ukuloba okuhlanekezelwe kanye nokukhulisa i-PCR, okuvumela i-reverse transcriptase ne-DNA polymerase ukuqedela ukusabela kushubhu efanayo ngaphansi kwezimo ezifanayo zebhafa.Isinyathelo esisodwa se-RT-qPCR sidinga kuphela ukusetshenziswa kweziqalo eziqondile zokulandelana.Ku-RT-qPCR yezinyathelo ezimbili, ukuloba okuhlanekezelwe kanye nokukhulisa i-PCR kwenziwa ngamashubhu amabili, kusetshenziswa izibhafa ezithuthukisiwe ezihlukene, izimo zokusabela, namasu omklamo wokuqala.
| Inzuzo | Ububi | |
| Isinyathelo esisodwa | Le ndlela inephutha elincane lokuhlola njengoba kokubili ukusabela kwenziwa ngeshubhu eyodwa
Izinyathelo ezimbalwa zamapayipi zinciphisa ingozi yokutheleleka
Ifanele ukukhuliswa okuphezulu/ukuhlolwa, kuyashesha futhi kukhiqizeka kabusha | Ukusabela okuyizinyathelo ezimbili akukwazi ukuthuthukiswa ngokuhlukana
Njengoba izimo zokusabela zifakwa engcupheni ngokuhlanganisa ukusabela kwezinyathelo ezimbili, ukuzwela akukuhle njengaleyo yezinyathelo ezimbili.
Inombolo yokuhlosiwe etholwe isampula eyodwa incane |
| Izinyathelo Ezimbili | Ikhono lokudala imitapo yolwazi ye-cDNA ezinzile engagcinwa isikhathi eside futhi isetshenziswe ekuphenduleni okuningi
Izakhi zofuzo eziqondiwe nezithenjwa zingandiswa kusukela kulabhulali efanayo ye-cDNA ngaphandle kwesidingo semitapo yolwazi eminingi ye-cDNA.
Izibhafa zokusabela nezimo zokusabela ezivumela ukulungiselelwa kokugijima kokusabela okukodwa
Ukukhetha okuguquguqukayo kwezimo zokucupha | Ukusebenzisa amashubhu amaningi, kanye nezinyathelo eziningi zamapayipi kwandisa ingozi yokungcoliswa kwe-DNA, futhi kuthatha isikhathi.
Idinga ukulungiselelwa okwengeziwe kunendlela yesinyathelo esisodwa |
Imikhiqizo ehlobene:
I-RT-qPCR Easyᵀᴹ (Isinyathelo esisodwa)-SYBR Green I
I-RT-qPCR Easyᵀᴹ (Isinyathelo Esisodwa)-Taqman
I-RT Easyᵀᴹ I Master Premix Ye-First-Strand CDNA Synthesis
Isikhathi Sangempela PCR Easyᵀᴹ-SYBR Green I Kit
Isikhathi Sangempela PCR Easyᵀᴹ-Taqman
Ukukhethwa kwesamba se-RNA ne-mRNA
Lapho udizayina isilingo se-RT-qPCR, kubalulekile ukunquma ukuthi usebenzise i-RNA ephelele noma i-mRNA ehlanzekile njengesifanekiso sokuhlehla okulotshiweyo.Nakuba i-mRNA ingase ikwazi ukunikeza ukuzwela okuphezulu kancane, i-RNA ephelele isasetshenziswa njalo.Isizathu salokhu ukuthi i-RNA ephelele inenzuzo ebaluleke kakhulu njengento yokuqala kune-mRNA.Okokuqala, inqubo idinga izinyathelo zokuhlanza ezimbalwa, eziqinisekisa ukutholwa komthamo okungcono kwesifanekiso kanye nokwenza ngcono kangcono imiphumela ukuze kuqalwe izinombolo zamaseli.Okwesibili, igwema isinyathelo sokucebisa i-mRNA, esingagwema amathuba okuba nemiphumela etshekile ngenxa yokutholwa okuhlukile kwama-mRNA ahlukahlukene.Sekukonke, njengoba ezinhlelweni eziningi ukulinganisa okuhlobene kofuzo oluqondiwe kubaluleke kakhulu kunokuzwela okuphelele kokutholwa, i-RNA ephelele ifaneleka kakhulu ezimweni eziningi.
Reverse transcription primer
Endleleni yezinyathelo ezimbili, izindlela ezintathu ezihlukene zingasetshenziswa ukuze kuqaliswe ukusabela kwe-cDNA: ama-oligo(dT) ama-primer, ama-primers angahleliwe, noma ama-primer aqondene nokulandelana kwawo.Ngokuvamile, ama-primers we-oligo(dT) kanye nama-primer angahleliwe asetshenziswa ngokuhlanganiswa.Lezi zingqalasizinda zigxila kucu lwesifanekiso se-mRNA futhi zinikeza i-reverse transcriptase nendawo yokuqala yokuhlanganisa.
| Ukukhetha kokuqala | Isakhiwo nomsebenzi | Inzuzo | Ububi |
| I-Oligo(dT) primer (noma i-oligo anchored (dT) primer) | Ukunwetshwa okunwetshiwe kwezinsalela ze-thymine ku-poly(A) umsila we-mRNA;i-anchor oligo(dT) primer iqukethe u-G, C, noma A ekugcineni kuka-3′ (isayithi lehange) | Ukuhlanganiswa kwe-cDNA yobude obugcwele kusuka ku-poly(A)-tailed mRNA
Isebenza uma kutholakala into yokuqala encane
Isizinda sokunamathisela siqinisekisa ukuthi i-oligo(dT) primer ibophezela ku-5′ poly(A) umsila we-mRNA. | Ifanele kuphela ukukhulisa izakhi zofuzo ezinemisila ye-poly(A).
Thola i-cDNA enqanyuliwe endaweni yokuqala*2 nge-poly(A)
Kuchemile ukubophezela kuze kube sekugcineni okungu-3*
*Lokhu kungenzeka kuncishiswe uma kusetshenziswa iziqalo ze-oligo(dT) eziqinile |
| i-primer engahleliwe
| 6 kuya ku-9 izisekelo ngobude, ezingaba kumasayithi amaningi ngesikhathi sokuloba kwe-RNA | I-Aneal kuwo wonke ama-RNA (tRNA, rRNA, kanye ne-mRNA)
Ifanele imibhalo enesakhiwo sesibili esibalulekile, noma uma kutholakala into yokuqala encane
Inani eliphakeme kakhulu lama-cDNA | I-cDNA ibhalwe ngokuhlehlayo ukusuka kuyo yonke i-RNA, ngokuvamile engathandeki futhi ingase inciphise isignali ye-mRNA eqondiwe.
thola i-cDNA encishisiwe |
| ama-primer aqondene nokulandelana | Iziqalo zangokwezifiso eziqondise ukulandelana okuqondile kwe-mRNA | umtapo wezincwadi we-cDNA othile
Thuthukisa ukuzwela
Isebenzisa iziqalo ze-qPCR ezihlehlayo | Kukhawulelwe kuphela ekuhlanganiseni kofuzo olulodwa oluqondiwe |
Reverse transcriptase
I-Reverse transcriptase iyi-enzyme esebenzisa i-RNA ukuze ihlanganise i-DNA.Amanye ama-reverse transcriptase anomsebenzi we-RNase futhi angehlisa isithunzi imicu ye-RNA kuma-RNA-DNA hybrid strands ngemva kokuloba.Uma ingenawo umsebenzi we-enzymatic we-RNase, i-RNaseH ingangezwa ngokusebenza kahle kwe-qPCR okuphezulu.Ama-enzyme asetshenziswa kakhulu ahlanganisa i-Moloney murine leukemia virus reverse transcriptase kanye ne-avian myeloblastoma virus reverse transcriptase.Ku-RT-qPCR, kuhle ukukhetha i-reverse transcriptase ene-thermostability ephakeme, ukuze ukuhlanganiswa kwe-cDNA kwenziwe emazingeni okushisa aphakeme, kuqinisekiswe ukulotshwa ngempumelelo kwama-RNA anesakhiwo sesibili esiphezulu, kuyilapho kugcinwa umsebenzi wawo ogcwele ngesikhathi sonke sokusabela, okuholela ekuvuthweni okuphezulu kwe-cDNA.
Imikhiqizo ehlobene:
I-Foreasy M-MLV Reverse Transcriptase
Umsebenzi we-RNase H we-reverse transcriptase
I-RNaseH iyakwazi ukuthunaza imicu ye-RNA isuka kumaduplex e-RNA-DNA, okuvumela ukuhlanganiswa okuphumelelayo kwe-DNA enemicu ekabili.Kodwa-ke, uma usebenzisa i-mRNA ende njengesifanekiso, i-RNA ingase yehliswe ngaphambi kwesikhathi, okuholela ekuncishisweni kwe-cDNA.Ngakho-ke, kuvame ukuzuzisa ukunciphisa umsebenzi we-RNaseH phakathi ne-cDNA cloning uma kufunwa ukuhlanganiswa kokulotshiwe okude.Ngokuphambene, okulotshiweyo okuhlanekezelwe okunomsebenzi we-RNase H kuvame ukuzuzisa izinhlelo zokusebenza ze-qPCR ngoba kuthuthukisa ukuncibilika kwamaduplex e-RNA-DNA phakathi nomjikelezo wokuqala we-PCR.
Idizayini yokuqala
Ama-primers e-PCR asetshenziselwa isinyathelo se-qPCR ku-RT-qPCR kufanele adizayinelwe ngendlela efanele ukuhlanganisa i-exon-exon, lapho isiqalo sokukhulisa amandla singase sidlule umngcele wangempela we-exon-intron.Njengoba ukulandelana kwe-genomic DNA equkethe i-intron kungakhuliswanga, lo mklamo wehlisa ubungozi bezinto ezingamanga ezikhuliswa ekungcoliseni i-genomic DNA.
Uma ama-primers angeke akhelwe ukuhlukanisa ama-exons noma imingcele ye-exon-exon, kungase kudingeke ukuthi uphathe amasampula e-RNA nge-RNase-free DNase I noma i-dsDNase ukuze kukhishwe ukungcoliswa kwe-genomic DNA.
Ukulawulwa kwe-RT-qPCR
Ukulawulwa okunegethivu kokulotshiweyo okuhlehlayo (ukulawula kwe-RT) kufanele kufakwe kukho konke ukuhlola kwe-RT-qPCR ukuze kutholwe ukungcola kwe-DNA (okufana ne-genomic DNA noma imikhiqizo ye-PCR kusukela ekuphenduleni kwangaphambilini).Lesi silawuli siqukethe zonke izingxenye zokusabela ngaphandle kwe-reverse transcriptase.Njengoba ukuloba okuhlanekezelwe kungenzeki nalokhu kulawulwa, uma ukukhuliswa kwe-PCR kubonwa, ukungcoliswa okuvela ku-DNA kungenzeka kakhulu.
Isikhathi sokuthumela: Aug-02-2022
