Izinto zokuqala: iRNA
Umyinge we-reverse transcription PCR (RT-qPCR) yindlela yovavanyo esetyenziswa kwimifuniselo ye-PCR kusetyenziswa i-RNA njengesixhobo sokuqala.Kule ndlela, i-RNA iyonke okanye i-RNA yesithunywa (mRNA) ibhalwa kuqala kwi-DNA ehambelanayo (cDNA) nge-reverse transcriptase.Emva koko, impendulo ye-qPCR yenziwa kusetyenziswa i-cDNA njenge template.I-RT-qPCR isetyenziswe kwiintlobo ngeentlobo zezicelo zebhayoloji ye-molekyuli, kubandakanywa uhlalutyo lwe-gene expression, ukuqinisekiswa kokuphazamiseka kwe-RNA, ukuqinisekiswa kwe-microarray, ukufumanisa i-pathogen, uvavanyo lwemfuzo, kunye nophando lwezifo.
Inyathelo elinye kunye neendlela ezimbini ze-RT-qPCR
I-RT-qPCR inokufezekiswa ngenyathelo elinye okanye indlela enamanyathelo amabini.Inyathelo elinye le-RT-qPCR lidibanisa ukubhalwa kwe-reverse kunye nokukhulisa i-PCR, ivumela i-reverse transcriptase kunye ne-DNA polymerase ukugqiba ukusabela kwi-tube efanayo phantsi kweemeko ezifanayo ze-buffer.Inyathelo elinye le-RT-qPCR lifuna kuphela ukusetyenziswa kweeprayimari ezithe ngqo ngolandelelwano.Kumanyathelo amabini e-RT-qPCR, ukukhutshelwa umva kunye nokwandiswa kwe-PCR kwenziwa kwiityhubhu ezimbini, kusetyenziswa ii-buffers ezahlukeneyo eziphuculweyo, iimeko zokusabela, kunye nezicwangciso zoyilo lwe-primer.
| Inzuzo | Ukusilela | |
| Inyathelo elinye | Le ndlela inemposiso encinci yovavanyo njengoko zombini iimpendulo zenziwa kwityhubhu enye
Amanyathelo emibhobho ambalwa anciphisa umngcipheko wosulelo
Ifanelekile kwi-high-throughput amplification/screening, fast and reproducible | Iimpendulo ezinamanyathelo amabini azinakwenziwa ngokwahlukeneyo
Ekubeni iimeko zokusabela ziphazamiseka ngokudibanisa amanyathelo amabini, uvakalelo alulunganga njengaleyo yeendlela ezimbini.
Inani leethagethi ezifunyenwe ngesampulu enye lincinci |
| Amanyathelo amabini | Ukukwazi ukwenza amathala eencwadi ecDNA azinzileyo anokugcinwa ixesha elide kwaye asetyenziswe kwiimpendulo ezininzi.
Iijeni ekujoliswe kuzo kunye nezalathiso zofuzo zinokunyuswa kwithala leencwadi elifanayo le-cDNA ngaphandle kwesidingo samathala eencwadi amaninzi e-cDNA.
Izithinteli zokusabela kunye neemeko zokusabela ezenza usebenziso lwendlela yokusabela enye
Ukukhetha okuguquguqukayo kweemeko zokuqalisa | Ukusebenzisa iityhubhu ezininzi, kunye namanyathelo ombhobho angakumbi anyusa umngcipheko wokungcoliseka kweDNA, kwaye kuthatha ixesha.
Ifuna ulungiselelo olungaphezulu kunendlela yenyathelo elinye |
Iimveliso eziyeleleneyo:
I-RT-qPCR Easyᵀᴹ (Inyathelo elinye)-SYBR Green I
RT-qPCR Easyᵀᴹ (Inyathelo elinye)-Taqman
RT Easyᵀᴹ I Master Premix Yeyokuqala-Strand CDNA Synthesis
Ixesha lokwenyani PCR Easyᵀᴹ-SYBR Green I Kit
Ixesha lokwenyani PCR Easyᵀᴹ-Taqman
Ukukhethwa kwe-RNA iyonke kunye ne-mRNA
Xa uyilwa umfuniselo we-RT-qPCR, kubalulekile ukwenza isigqibo sokuba usebenzise i-RNA iyonke okanye i-mRNA esulungekileyo njengethempleyithi yokukhutshelwa umva.Nangona i-mRNA inokukwazi ukubonelela ngovakalelo oluphezulu kancinci, iyonke i-RNA isasetyenziswa rhoqo.Isizathu soku kukuba i-RNA iyonke inenzuzo ebaluleke ngakumbi njengesixhobo sokuqala kune-mRNA.Okokuqala, le nkqubo idinga amanyathelo okuhlanjululwa okumbalwa, okuqinisekisa ukubuyiswa kobungakanani obungcono be-template kunye nokuqhelaniswa okungcono kweziphumo zokuqalisa iinombolo zeeseli.Okwesibini, ithintela inyathelo lokutyebisa le-mRNA, elinokuthi lithintele ukubakho kweziphumo ezigqwesileyo ngenxa yokufunyanwa kwakhona kwe-mRNAs ezahlukeneyo.Ngokubanzi, kuba kwizicelo ezininzi ubungakanani obunxulumene nejini ekujoliswe kuyo ibaluleke ngakumbi kunovakalelo olupheleleyo lokubhaqwa, iyonke i-RNA ifaneleke ngakumbi kwiimeko ezininzi.
Reverse transcription primer
Kwindlela yamanyathelo amabini, iindlela ezintathu ezahlukeneyo zingasetyenziselwa ukukhupha i-cDNA reaction: i-oligo (dT) i-primers, i-random primers, okanye i-primers echanekileyo yokulandelelana.Ngokuqhelekileyo, iiprimers ze-oligo (dT) kunye ne-random primers zisetyenziswa ngokudibeneyo.Ezi primers anneal kwi-template mRNA strand kwaye zibonelela nge-reverse transcriptase kunye nesiqalo se-synthesis.
| Ukhetho lokuqala | Ulwakhiwo kunye nomsebenzi | Inzuzo | Ukusilela |
| I-Oligo(dT) primer (okanye i-oligo anchored (dT) primer) | Ukwandiswa kwe-annealing kwiintsalela ze-thymine kumsila we-poly(A) we-mRNA;i-anchor oligo(dT) primer iqulethe i-G, C, okanye A kwisiphelo se-3′ (indawo yeankile) | Ukuhlanganiswa kwe-cDNA yobude obupheleleyo ukusuka kwi-poly(A)-tailed mRNA
Isebenza xa kufumaneka imathiriyeli yokuqala encinane
Indawo ye-ankile iqinisekisa ukuba i-oligo(dT) primer ibophelela kwi-5′ poly(A) umsila we-mRNA. | Ifanele kuphela ukukhulisa imfuza enemisila yepoly(A).
Fumana i-cDNA ecuthiweyo kwindawo yokuqala*2 kwipoly(A)
Kuthambekele ekubopheleleni ukuya esiphelweni sesi-3*
*Oku kunokwenzeka kuncitshiswe ukuba iiprimer ze-oligo(dT) ezizinzisiweyo ziyasetyenziswa |
| primer random
| Iziseko ezi-6 ukuya kwezisi-9 ngobude, ezinokungena kwiindawo ezininzi ngexesha loshicilelo lwe-RNA | IAneal kuzo zonke ii-RNAs (tRNA, rRNA, kunye ne-mRNA)
Ilungele imibhalo enesakhiwo esibalulekileyo sesibini, okanye xa kufumaneka imathiriyeli yokuqala encinane
Isivuno esiphezulu se-cDNA | I-cDNA iguqulelwa umva kuyo yonke i-RNA, ehlala inganqweneleki kwaye inokunciphisa umqondiso we-mRNA ekujoliswe kuyo.
fumana i-cDNA encinci |
| iiprimers ezilandelelanayo | Iiprimers eziqhelekileyo ezijolise kulandelelwano oluthile lwe-mRNA | ilayibrari ethile ye-cDNA
Phucula ubuntununtunu
Ukusebenzisa iiprayimari ze-qPCR ezibuyela umva | Kuphela kuthintelwe kuqulunqo lwejini enye ekujoliswe kuyo |
Reverse transcriptase
I-reverse transcriptase yi-enzyme esebenzisa i-RNA ukudibanisa i-DNA.Ezinye ii-reverse transcriptases zinomsebenzi we-RNase kwaye zinokuthoba imicu ye-RNA kwi-RNA-DNA hybrid strands emva koshicilelo.Ukuba ayinawo umsebenzi we-enzymatic ye-RNase, i-RNaseH inokongezwa kubuchule obuphezulu be-qPCR.Ii-enzymes ezisetyenziswa ngokuqhelekileyo ziquka i-Moloney murine leukemia virus reverse transcriptase kunye ne-avian myeloblastoma virus reverse transcriptase.Kwi-RT-qPCR, kulungele ukukhetha i-reverse transcriptase ene-thermostability ephezulu, ukwenzela ukuba i-cDNA synthesis inokwenziwa kumaqondo obushushu aphezulu, ukuqinisekisa ukubhaliswa okuyimpumelelo kwe-RNAs kunye nesakhiwo sesibini esiphezulu, ngelixa igcina umsebenzi wayo opheleleyo ngexesha lonke lokuphendula, okukhokelela kwimveliso ephezulu ye-cDNA.
Iimveliso eziyeleleneyo:
I-M-MLV Reverse Transcriptase ilula
Umsebenzi we-RNase H we-reverse transcriptase
I-RNaseH iyakwazi ukuthobisa imicu ye-RNA ukusuka kwi-RNA-DNA duplexes, evumela ukuhlanganiswa okusebenzayo kwe-DNA ephindwe kabini.Nangona kunjalo, xa usebenzisa i-mRNA ende njenge template, i-RNA inokuthotywa kwangaphambi kwexesha, okukhokelela kwi-cDNA ecuthiweyo.Ke ngoko, kuhlala kuba luncedo ukunciphisa umsebenzi we-RNaseH ngexesha le-cDNA cloning ukuba udityaniso lwemibhalo emide iyafunwa.Ngokwahlukileyo koko, ukubuyisela umva i-transcriptases kunye nomsebenzi we-RNase H zihlala ziluncedo kwizicelo ze-qPCR kuba ziphucula ukunyibilika kwe-RNA-DNA duplexes ngexesha lomjikelo wokuqala we-PCR.
Uyilo lokuqala
Iiprayimari ze-PCR ezisetyenziselwa inyathelo le-qPCR kwi-RT-qPCR kufuneka ziyilwe ngokufanelekileyo ukuba zivule isiphambuka se-exon-exon, apho i-primer yokukhulisa inokubanakho ukolula umda wokwenyani we-exon-intron.Ekubeni i-intron-containing genomic DNA sequences ayinyuswanga, olu yilo lunciphisa umngcipheko weengxelo zobuxoki ezikhuliswe ekungcoliseni i-DNA ye-genomic.
Ukuba iiprimers azinakuyilwa ukwahlula i-exons okanye imida ye-exon-exon, kunokuba yimfuneko ukunyanga iisampulu ze-RNA nge-RNAse-free DNase I okanye i-dsDNase ukususa ukungcoliseka kwe-genomic DNA.
Ulawulo lwe-RT-qPCR
Ulawulo olungalunganga loshicilelo olubuyisela umva (-ulawulo lwe-RT) kufuneka luqukwe kuzo zonke iimfuniselo ze-RT-qPCR zokubona ungcoliseko lwe-DNA (njenge-genomic DNA okanye iimveliso ze-PCR ezivela kwiimpendulo zangaphambili).Olu lawulo luqulathe onke amalungu empendulo ngaphandle kwe-reverse transcriptase.Ekubeni ukukhutshelwa umva akwenzeki ngolu lawulo, ukuba i-PCR yokukhulisa ibonwa, ungcoliseko oluvela kwi-DNA lunokwenzeka kakhulu.
Ixesha lokuposa: Aug-02-2022
