Kutanga zvinhu: RNA
Quantitative reverse transcription PCR (RT-qPCR) inzira yekuyedza inoshandiswa mukuyedza PCR uchishandisa RNA sechinhu chekutanga.Munzira iyi, RNA yese kana mutumwa RNA (mRNA) inotanga kunyorwa mumubatanidzwa weDNA (cDNA) nereverse transcriptase.Zvadaro, qPCR reaction yakaitwa uchishandisa cDNA setemplate.RT-qPCR yakashandiswa mumhando dzakasiyana dzemamolecular biology maapplication, kusanganisira gene expression analysis, RNA interference validation, microarray validation, pathogen kuonekwa, genetic test, uye tsvakurudzo yechirwere.
Imwe-nhanho uye mbiri-nhanho nzira dzeRT-qPCR
RT-qPCR inogona kuitwa nedanho-rimwe kana maviri-nhanho nzira.Imwe-nhanho RT-qPCR inosanganisa reverse transcription uye PCR amplification, ichibvumira reverse transcriptase uye DNA polymerase kupedzisa kuita muchubhu imwechete pasi pemamiriro akafanana ebuffer.Imwe-nhanho RT-qPCR inongoda chete kushandiswa kwekutevedzana-chaiwo maprimers.Mune maviri-nhanho RT-qPCR, reverse transcription uye PCR amplification inoitwa mumachubhu maviri, uchishandisa akasiyana optimized buffers, maitiro ekuita, uye primer dhizaini mazano.
| Advantage | Kukanganisa | |
| Imwe Danho | Iyi nzira ine chikanganiso chidiki chekuyedza sezvo zvese zvinobatika zvichiitwa muchubhu imwe
Matanho mashoma epaipi anoderedza njodzi yekusvibiswa
Inokodzera yakakwirira-kuburikidza amplification / kuongorora, inokurumidza uye inogadzirwazve | Matanho maviri ekuita haagone kugadziridzwa zvakasiyana
Sezvo mamiriro ekuita achikanganiswa nekubatanidza maitiro maviri-nhanho, kunzwisiswa hakuna kunaka seye nzira mbiri-nhanho.
Nhamba yezvinangwa zvakaonekwa nemuenzaniso mumwechete idiki |
| Matanho maviri | Kugona kugadzira yakagadzika cDNA maraibhurari anogona kuchengetwa kwenguva yakareba uye kushandiswa mukuita kwakawanda.
Target majini uye mareferenzi genes anogona kukwidziridzwa kubva kune imwechete cDNA raibhurari pasina kudiwa kweakawanda cDNA maraibhurari.
Reaction buffers uye maitiro ekuita anogonesa optimization yeimwe reaction inomhanya
Flexible sarudzo ye trigger mamiriro | Kushandisa akawanda machubhu, uye mamwe nhanho yekupopa inowedzera njodzi yekusvibiswa kweDNA, uye zvinotora nguva.
Inoda kuwedzera optimization pane imwe-danho nzira |
Zvigadzirwa zvinoenderana:
RT-qPCR Easyᵀᴹ (Imwe Danho) -SYBR Green I
RT-qPCR Easyᵀᴹ (Imwe Danho)-Taqman
RT Easyᵀᴹ I Master Premix Yekutanga-Strand CDNA Synthesis
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Kusarudzwa kweiyo RNA yakazara uye mRNA
Paunenge uchigadzira kuyedza kweRT-qPCR, zvakakosha kuti usarudze kuti woshandisa yakazara RNA here kana yakacheneswa mRNA setemplate yekudzosera kumashure.Kunyangwe mRNA ichikwanisa kupa kunzwisiswa kwepamusoro zvishoma, yakazara RNA ichiri kushandiswa kazhinji.Chikonzero cheizvi ndechekuti RNA yakazara ine mukana wakanyanya kukosha sechinhu chekutanga kupfuura mRNA.Chekutanga, maitiro acho anoda mashoma ekucheneswa matanho, ayo anovimbisa zvirinani kuwanda kudzoreredza kwetemplate uye zvirinani kudzoreredza mhedzisiro yekutanga nhamba dzesero.Chechipiri, inodzivirira mRNA nhanho yekupfumisa, iyo inogona kudzivirira mukana wekumisikidzwa mhedzisiro nekuda kwekudzoreredzwa kwakasiyana kwemRNAs dzakasiyana.Pakazere, sezvo mumashandisirwo mazhinji chiyero chehukama chejini chinotarirwa chakanyanya kukosha pane kunzwisiswa kwekuona, yakazara RNA inonyanya kukodzera muzviitiko zvakawanda.
Reverse transcription primer
Munzira mbiri-nhanho, nzira nhatu dzakasiyana dzinogona kushandiswa kuprimisa cDNA reaction: oligo(dT) primers, random primers, kana kutevedzana-chaiyo primers.Kazhinji, oligo(dT) maprimers uye zvisingaite maprimers anoshandiswa musanganiswa.Aya maprimers anneal kune template mRNA strand uye anopa reverse transcriptase nenzvimbo yekutanga yekubatanidza.
| Kusarudzwa kwekutanga | Chimiro uye basa | Advantage | Kukanganisa |
| Oligo(dT) primer (kana anchored oligo(dT) primer) | Yakawedzerwa annealing kune thymine zvakasara pa poly(A) muswe wemRNA;anchor oligo(dT) primer ine G, C, kana A kumagumo 3′ (ancho saiti) | Synthesis yehurefu hwakazara cDNA kubva papoly(A) -tailed mRNA
Inoshanda kana zvinhu zvishoma zvekutanga zviripo
Anchoring saiti inovimbisa kuti oligo(dT) primer inosunga kune 5′ poly(A) muswe wemRNA. | Inokodzera chete kukwirisa majini ane poly(A) miswe
Tora cDNA yakatemwa kubva panzvimbo yekutanga * 2 mu poly(A)
Yakarerekera kusunga kusvika kumagumo e3*
*Kugona uku kunodzikiswa kana anchored oligo(dT) primers akashandiswa |
| random primer
| 6 kusvika 9 mabhesi pakureba, ayo anogona kubatanidza kune akawanda masayiti panguva yekunyorwa kweRNA | Anneal kune ese maRNAs (tRNA, rRNA, uye mRNA)
Inokodzera zvinyorwa zvine kukosha kwechipiri chimiro, kana kana zvishoma zvekutanga zviripo
Yakakwira cDNA Goho | cDNA inodzokororwa yakanyorwa kubva kune yese RNA, iyo isingawanzo kudiwa uye inogona kuderedza chiratidzo chechinangwa mRNA.
kuwana cDNA truncated |
| kutevedzana-chaiyo primers | Tsika primers yakanangana nemRNA yakatevedzana | chaiyo cDNA raibhurari
Vandudza kunzwa
Kushandisa reverse qPCR primers | Inongogumira pakubatanidzwa kwejini rimwechete rinotarirwa |
Reverse transcriptase
Reverse transcriptase ienzyme inoshandisa RNA kugadzira DNA.Mamwe mareverse transcriptase ane chiitiko cheRNase uye anogona kukanganisa RNA tambo muRNA-DNA hybrid tambo mushure mekunyorwa.Kana isina RNase enzymatic chiitiko, RNaseH inogona kuwedzerwa kwepamusoro qPCR kunyatsoshanda.Ma enzymes anowanzo shandiswa anosanganisira Moloney murine leukemia virus reverse transcriptase uye avian myeloblastoma virus reverse transcriptase.Kune RT-qPCR, zvakanakira kusarudza reverse transcriptase ine yakakwirira thermostability, kuitira kuti cDNA synthesis iitwe patembiricha yepamusoro, kuve nechokwadi chekubudirira kunyorwa kweRNAs nepamusoro sekondari chimiro, uku ichichengeta yavo yakazara basa mukati mekuita, zvichikonzera kukwira kwe cDNA goho.
Zvigadzirwa zvinoenderana:
Foreasy M-MLV Reverse Transcriptase
RNase H chiitiko che reverse transcriptase
RNaseH inokwanisa kushatisa RNA tambo kubva kuRNA-DNA duplexes, ichibvumira kuumbwa kwakanaka kweDNA ine tambo mbiri.Nekudaro, kana uchishandisa refu mRNA setemplate, iyo RNA inogona kuderedzwa nguva isati yakwana, zvichikonzera truncated cDNA.Naizvozvo, zvinowanzobatsira kudzikisira chiitiko cheRNaseH panguva yecDNA cloning kana synthesis yezvinyorwa zvirefu zvichidikanwa.Mukupesana, reverse transcriptases ane RNase H chiitiko anowanzo kubatsira qPCR maapplication nekuti anosimudzira kunyungudika kweRNA-DNA duplexes panguva yekutanga kutenderera kwePCR.
Primer design
PCR maprimers anoshandiswa qPCR nhanho muRT-qPCR inofanirwa kugadzirwa kuti itore exon-exon junction, apo inosimudzira primer inogona kutenderedza exon-intron muganhu.Sezvo intron-ine genomic DNA sequences haina kukwidziridzwa, dhizaini iyi inoderedza njodzi yenhema inowedzerwa kubva mukusvibisa genomic DNA.
Kana maprimers asingakwanise kugadzirwa kuti aparadzanise maexons kana exon-exon miganhu, zvingave zvakafanira kurapa maRNA samples neRNase-isina DNase I kana dsDNase kubvisa genomic DNA kusvibiswa.
RT-qPCR kutonga
A reverse transcription negative control (-RT control) inofanira kuverengerwa mune zvese RT-qPCR kuyedza kuona kusvibiswa kweDNA (senge genomic DNA kana PCR zvigadzirwa kubva pane zvakamboitika).Kudzora uku kune zvese zvinobatika kunze kwekunge reverse transcriptase.Sezvo reverse transcription isingaitiki nekutonga uku, kana PCR amplification ikaonekwa, kusvibiswa kubva kuDNA kunowanzoitika.
Nguva yekutumira: Aug-02-2022
