Waxyaabaha laga bilaabo: RNA
Tijaabada dib-u-celinta tirada PCR (RT-qPCR) waa hab tijaabo ah oo lagu isticmaalo tijaabooyinka PCR iyadoo la isticmaalayo RNA oo ah walxo bilow ah.Habkan, wadarta guud ee RNA ama Messenger RNA (mRNA) ayaa marka hore loo qoraa DNA dhammaystiran (cDNA) iyada oo loo marayo transcriptase.Ka dib, falcelinta qPCR ayaa la sameeyay iyadoo la isticmaalayo cDNA qaab ahaan.RT-qPCR waxaa loo adeegsaday noocyo kala duwan oo ah codsiyada bayoloji molecular, oo ay ku jiraan falanqaynta muujinta hidda-socodka, ansaxinta faragelinta RNA, xaqiijinta microarray, ogaanshaha cudur-sidaha, baaritaanka hidda-socodka, iyo cilmi-baarista cudurrada.
Hal-tallaabo iyo hababka laba-tilaabo ee RT-qPCR
RT-qPCR waxaa lagu fulin karaa hal-tallaabo ama hab laba-tallaabo ah.Hal-tallaabo RT-qPCR waxay isku daraysaa qoraal-celinta iyo kordhinta PCR, taas oo u oggolaanaysa transcriptase-ka-noqoshada iyo polymerase DNA inay dhammaystirto fal-celinta isla tuubo isku mid ah oo hoos yimaada isla shuruudaha kaydinta.Hal-tallaabo RT-qPCR waxay u baahan tahay oo kaliya isticmaalka furayaasha gaarka ah ee isku xigxiga.Laba-tallaabo ee RT-qPCR, qoraal-ku-noqosho iyo kordhinta PCR ayaa lagu sameeyaa laba tuubbo, iyadoo la adeegsanayo kaydin la hagaajiyay oo kala duwan, xaaladaha falcelinta, iyo xeeladaha naqshadaynta aasaasiga ah.
| Faa'iidada | Khasaaro | |
| Hal Tilaabo | Habkani waxa uu leeyahay khalad tijaabo ah oo yar maadaama labada falcelin lagu sameeyo hal tuubo
Tillaabooyinka tuubooyinka oo yaraada waxay yareeyaan khatarta wasakheynta
Ku habboon kor-u-qaadis/baaritaan-samayn-sare leh, degdeg ah oo dib loo soo saari karo | Falcelinta laba-tallaabo si gaar ah looma hagaajin karo
Mar haddii xaaladaha falcelintu ay wax u dhimeen iyadoo la isku darayo falcelinta laba-tallaabo ah, dareenku uma fiicna sida habka laba-tallaabo.
Tirada bartilmaameedyada lagu ogaaday hal muunad ayaa yar |
| Laba Tallaabo | Awoodda lagu abuuro maktabado cDNA ah oo deggan oo la kaydin karo waqti dheer loona isticmaalo falcelinno badan
Hidde-sidaha bartilmaameedka ah iyo hiddo-wadaha tixraaca ayaa laga kordhin karaa isla maktabadda cDNA iyada oo aan loo baahnayn maktabado badan oo cDNA ah
Xakamaynta falcelinta iyo xaaladaha falcelinta ee suurtageliya hagaajinta falcelinta hal mar
Xulashada dabacsan ee xaaladaha kiciya | Isticmaalka tubooyinka badan, iyo tillaabooyin badan oo tuubooyinka waxay kordhiyaan halista wasakhowga DNA-da, iyo waqti badan.
Waxay u baahan tahay hagaajin ka badan habka hal-tallaabo |
Alaabta la xidhiidha:
RT-qPCR Easyᵀᴹ (Hal Talaabo) -SYBR Cagaaran I
RT-qPCR Easyᵀᴹ (Hal Talaabo) -Taqman
RT Easyᵀᴹ I Master Premix ee Xarig-koowaad ee CDNA Synthesis
Waqtiga dhabta ah PCR Easyᵀᴹ-SYBR Cagaarka I Kit
Waqtiga dhabta ah PCR Easyᵀᴹ-Taqman
Xulashada wadarta RNA iyo mRNA
Marka la naqshadeynayo tijaabada RT-qPCR, waxaa muhiim ah in la go'aansado in la isticmaalo wadarta RNA ama mRNA la safeeyey sidii qaab-qoraal celin ah.In kasta oo mRNA laga yaabo inay bixiso dareen waxyar ka sarreeya, wadarta guud ee RNA ayaa wali la isticmaalaa.Sababta tani waa in wadarta guud ee RNA ay leedahay faa'iido aad u muhiim ah sida walxaha bilawga ah marka loo eego mRNA.Marka hore, geeddi-socodku wuxuu u baahan yahay tillaabooyin nadiifin oo ka yar, taasoo hubinaysa soo kabashada tirada wanaagsan ee qaab-dhismeedka iyo caadiyeynta natiijooyinka si loo bilaabo tirooyinka unugyada.Marka labaad, waxay ka fogaataa tallaabada kobcinta mRNA, taas oo ka fogaan karta suurtogalnimada natiijooyinka qalloocan sababtoo ah soo kabashada kala duwan ee mRNA-yada kala duwan.Guud ahaan, maadaama codsiyada intooda badan qiyaasta qiyaasta hidde-sidaha bartilmaameedku ay aad uga muhiimsan tahay dareenka saxda ah ee ogaanshaha, wadarta guud ee RNA ayaa ku habboon xaaladaha badankood.
Dib-u-celinta qoraalka
Habka laba-tallaabo, saddex hab oo kala duwan ayaa loo isticmaali karaa in lagu hormariyo fal-celinta cDNA: oligo(dT) aasaasayaasha, curiyeyaasha random-ka, ama aasaasayaasha u gaarka ah taxanaha.Caadi ahaan, oligo(dT) furayaasha iyo furayaasha random-ka ayaa la istcimalaa marka la isku daro.Aasaasayaashani waxay ku sii durkayaan qaab-dhismeedka mRNA-ga oo waxay bixiyaan qoraal-ku-soo-noqod oo leh barta bilawga ah ee isku-dhafka.
| Xulashada koowaad | Qaab dhismeedka iyo shaqada | Faa'iidada | Khasaaro |
| Oligo(dT) furaha (ama oligo(dT) furaha ku xidhan) | Sii fidsan ee hadhaaga thymine ee dabada poli(A) ee mRNA;barroosin oligo(dT) ka kooban yahay G, C, ama A ee dhamaadka 3′ (goobta barroosinka) | Isku-dubaridka cDNA-dherer buuxa oo ka yimid poly(A) -mRNA
Lagu dabaqi karo marka wax yar la heli karo
Meesha ku xidhiddu waxay hubisaa in oligo(dT) asaasiga ahi ku xidho 5' dabada (A) ee mRNA | Kaliya waxay ku habboon tahay in lagu kordhiyo hiddo-wadaha leh dabada poly(A).
Hel cDNA oo laga gooyay goobta hore*2 ee poly(A)
U xaglin in lagu xidho dhammaadka 3′*
* Suurtagalnimadan waa la yareeyaa haddii la isticmaalo aasaasayaasha oligo(dT). |
| asal random
| 6 ilaa 9 saldhig oo dherer ah, kaas oo soo saari kara goobo badan inta lagu jiro qoraalka RNA | Anneal dhammaan RNA-yada (tRNA, rRNA, iyo mRNA)
Ku haboon qoraalada leh qaab dhismeed sare oo muhiim ah, ama marka wax bilow ah la heli karo
Waxsoosaarka cDNA sare | cDNA waa laga soo rogay dhammaan RNA, taas oo inta badan aan la rabin oo laga yaabaa inay khafiifiso calaamadda mRNA bartilmaameedka.
la googooyo cDNA |
| sooyaal-gaar ah oo taxane ah | Aasaaska gaarka ah ee beegsanaya taxanaha mRNA ee gaarka ah | maktabad cDNA gaar ah
Hagaajinta dareenka
Isticmaalka qormayaasha qPCR gadaal | Kaliya waxay ku xaddidan tahay isku-dhafka hal hidde-siyeedka |
Gadaal qoraalka
Reverse transcriptase waa enzim u isticmaala RNA si uu u soo saaro DNA.Qaar ka mid ah qoraalada dib-u-celinta waxay leeyihiin waxqabad RNase waxayna hoos u dhigi karaan xargaha RNA ee xargaha isku-dhafka ah ee RNA-DNA ka dib qoraalka.Haddii aysan lahayn dhaqdhaqaaqa enzymatic RNase, RNaseH waxaa lagu dari karaa waxtarka sare ee qPCR.Ensaymes-yada sida caadiga ah loo isticmaalo waxaa ka mid ah Moloney murine leukemia fayraska reverse transcriptase iyo fayraska myeloblastoma reverse transcriptase.RT-qPCR, waxay ku habboon tahay in la doorto qoraal-ku-celin ah oo leh heerkul sarreeya, si isku-darka cDNA lagu samayn karo heerkul sare, hubinta in si guul leh loo qoro RNA-yada qaab dhismeed sare sare leh, iyada oo la ilaalinayo dhaqdhaqaaqooda buuxa inta lagu jiro falcelinta, taasoo keentay wax-soo-saarka cDNA sare.
Alaabta la xidhiidha:
Horudhac M-MLV ah Transcriptase
Dhaqdhaqaaqa RNase H ee qoraalka qoraalka ah
RNaseH waxay awood u leedahay inay hoos u dhigto xargaha RNA ee duplexes-ka RNA-DNA, taasoo u oggolaanaysa in si hufan loo sameeyo DNA-da laba-geesoon.Si kastaba ha ahaatee, marka loo isticmaalo mRNA dheer qaab template ah, RNA waxaa laga yaabaa in ay hoos u dhac ku yimid, taasoo keentay in cDNA googo'an.Sidaa darteed, inta badan waa faa'iido in la yareeyo dhaqdhaqaaqa RNaseH inta lagu jiro cloning cDNA haddii la rabo qoraallada dheer.Taas bedelkeeda, qoraalada dib u noqoshada ee waxqabadka RNase H ayaa inta badan faa'iido u leh codsiyada qPCR sababtoo ah waxay kor u qaadaan dhalaalka RNA-DNA duplexes inta lagu jiro wareegga koowaad ee PCR.
Naqshadaynta asaasiga ah
Qorshayaasha PCR ee loo isticmaalo tallaabada qPCR ee RT-qPCR waa in sida ugu habboon loo qaabeeyey in lagu fidiyo isgoysyada exon-exon, halkaas oo furaha cod-weyneyuhu uu awood u leeyahay inuu kordho xuduud dhab ah oo exon-intron ah.Mar haddii taxanaha DNA-da intron-ku-jira aan la kordhin, nashqadani waxay yaraynaysaa khatarta been abuurka ah ee laga soo qaaday wasakhaynta DNA-da.
Haddii aasaasayaasha aan la naqshadayn karin si loo kala saaro exons ama xuduudaha exon-exon, waxaa laga yaabaa inay lagama maarmaan noqoto in lagu daweeyo muunada RNA DNA-ga ka xora ah DNase I ama dsDNase si meesha looga saaro wasakheynta DNA-da.
Xakamaynta RT-qPCR
Xakamaynta diidmada qoraalka gadaasha (-RT) waa in lagu daraa dhammaan tijaabooyinka RT-qPCR si loo ogaado wasakhowga DNA (sida DNA-da genomic ama PCR ee falcelinta hore).Xakamayntani waxay ka kooban tahay dhammaan qaybaha falcelinta marka laga reebo qoraal-ku-soo-noqoshada.Mar haddii qoraalka gadaasha uusan ku dhicin kontoroolkan, haddii kor u qaadista PCR la arko, faddaraynta DNA-da waxay u badan tahay.
Waqtiga boostada: Agoosto-02-2022
