RT-qPCR kuyedza inosanganisira RNA kudhirowa uye kuongororwa kwemhando, reverse transcription uye qPCR matanho matatu, nhanho imwe neimwe ine zvakawanda zvekuchengetedza, isu tichazivisa zvakadzama pazasi.

Ⅰ.RNA yemhando yekuongorora

Mukuedza kweRT-qPCR, mushure mekupedzwa kweRNA kudhirowa, mhando yeRNA inoda kuongororwa, uye kuyedza kwekutevera kunogona kuitwa chete mushure mekukodzera.Nzira dzekuongorora dzinosanganisira spectrophotometer, Agilent gel electrophoresis, Agilent 2100 kuongorora, pakati peinonyanya kushandiswa spectrophotometer uye agarose gel electrophoresis nzira yekuongorora.Zvinofanira kucherechedzwa kuti nzira mbiri idzi dzinoda kushandiswa pamwe chete kupedzisa kuongororwa uye kuongororwa kweRNA kusungwa, kuchena uye kutendeseka, kuitira kuve nechokwadi chemhando yeRNA.

Inoenderana RNA Isolation Kit: 

Sero Yese RNA Isolation Kit

Yakanyanya kucheneswa uye yemhando yepamusoro RNA inogona kuwanikwa kubva kune akasiyana siyana maseru mu11min.

Mhuka Yese RNA Isolation Kit

Nekukurumidza uye zvinobudirira bvisa yakakwira-kuchena uye yemhando yepamusoro RNA kubva kune akasiyana mhuka tishu.

Spectrophotometer:

Iyo spectrophotometer inonyanya kushandiswa kuona kusungirirwa uye kuchena kweRNA, asi haigone kuona kutendeseka kweRNA uye genomic zvakasara.Pakati pavo, A260/280 uye A260/230 ndiwo akakosha paramita ekuonekwa kweRNA kuchena, uye kuchena kweRNA kunogona kuwonekwa zvichienderana nekushanduka kwehunhu hwavo:

1. 1.9< A260/280< 2.1, zvichiratidza kuti kuchena kweRNA kwakanaka;A260/280<1.9, zvichiratidza kuti panogona kunge paine mapuroteni akasara muRNA;A260/280> 2.1, zvichiratidza kuti zvinogona kukanganisa zvishoma zveRNA, izvo zvinogona kusimbiswa zvakare neagarose gel electrophoresis.

2. 2.0< A260/230< 2.2, zvichiratidza kuti kuchena kweRNA kwakanaka;A260/230< 2.0, zvichiratidza kuti panogona kunge paine zvakasara zveorganic reagents muRNA, senge phenols, ethanol kana shuga.

Agarose gel electrophoresis:

Agarose gel electrophoresis assay inogona kuongorora RNA kutendeseka, genome uye mapuroteni masara, asi haigone kunyatso kuenzanisa kuunganidzwa kweRNA kana kuona zvakasara zveorganic reagents.Tora eukaryotic RNA matemplate semuenzaniso:

1. Iyo RNA yaive pasi peagarose gel electrophoresis.Kana paingova nemabhendi matatu chete e28sRNA, 18sRNA uye 5.8sRNA pamepu yegel, zvinoratidza kuti RNA yakabviswa iripo.Kana pane chiitiko chinokweva, chinoratidza kuderera kweRNA.

2. Kana pane bhandi rimwe chete rakajeka pakati pegomba reglue uye 28sRNA band, panogona kunge kune genomic DNA yakasara.

3. Kana mabhendi achionekwa mugomba reglue, zvinoratidza kuti panogona kunge kune zvakasara zveprotein uye mamwe macromolecular substances.

Ⅱ. Reverse transcription

Mushure mekubviswa kweRNA kwapera, inoda kudzoserwa kuita cDNA yezviedzo zvinotevera, saka danho rekudzosera rakakosha.Reverse transcription ichaunzwa kubva pakusarudza reverse transcriptase uye primer:

Reverse transcriptase sarudzo:

Iyo yakajairika reverse transcriptases inosanganisira AMV RTase uye MMLV RTase.Iyo RNase H ye AMV RTase ine chiitiko chakasimba, pfupi synthesis urefu, yakaderera synthesis huwandu uye yakanaka kugadzikana kwekupisa (42 ~ 55 ℃).Iyo RNase H chiitiko cheMMLV RTase haina kusimba, iyo synthesis urefu hwakareba, iyo synthesis huwandu hwakakwira, uye kugadzikana kwekupisa kwakashata (37 ~ 42 ℃).

Nekuti RNase H enzyme ine basa rekusvibisa RNA template, MMLV ine isina simba RNase H basa rinofanirwa kusarudzwa panguva yereverse transcription, uye mushure mekupedzisira genetic engineering, kugadzikana kwekupisa kweMMLV kwasvika pakusvetuka kwehunhu.Kutora ForegeneForeasy Reverse Transcriptase(M-MLV yereverse transcription) semuenzaniso, itsva reverse transcriptase inoratidzwa muE. coli engineered mabhakitiriya achishandisa genetic recombination technology.Iyo inodzokorodza DNA polymerase iyo inogadzira inowirirana DNA strand kubva kune imwe-stranded RNA, DNA, kana RNA: DNA hybrid.Iyo haina RNase H chiitiko, kugadzikana kwakasimba, yakasimba RNA affinity, uye yakakwirira yekuona senitivity.

 

Foreasy Reverse Transcriptase(M-MLV yereverse transcription)

Kusarudzwa kwekutanga:

Kazhinji RT primers inowira mumapoka matatu: oligo dT, random primers, uye gene-chaiyo primers.Sarudza maprimers akakodzera ekushandisa zvinoenderana neakasiyana ekuedza zvinodiwa.

1. Kana template iri eukaryotic mavambo uye cDNA yakanonoka inoshandiswa muitiro PCR amplification, Oligo (dT) inokurudzirwa;Kana kuyedza kunotevera kuchishandiswa qPCR chete, Oligo (dT) inokurudzirwa kuti isanganiswe nemaprimers asina kurongeka kuti ivandudze mashandiro ereverse transcription.

2. Kana template iri kubva kuprokaryotes, Random Primers kana gene specific primers inofanira kusarudzwa kuti inyore reverse.

Ⅲ.qPCR

Fluorescence quantification inonyanya kutsanangurwa kubva pakusarudzwa kwehuwandu nzira, primer dhizaini misimboti, ROX kusarudzwa, reaction system kumisikidza uye maitiro ekugadzirisa mamiriro, nezvimwe.

Kusarudzwa kwehuwandu nzira:

Quantitative nzira dzakakamurwa kuva hukama hwehuwandu nzira uye yakakwana yehuwandu nzira.Relative quantification inogona kushandiswa kuona mhedzisiro yedzimwe nzira dzekurapa pakutaura kwejeni, kuona mutsauko wekutaura kwemajini panguva dzakasiyana uye kuenzanisa mutsauko wekutaura kwejeni mumatishu akasiyana.Absolute quantification inogona kuona huwandu hwenyucleic acid muhutachiona uye zvichingodaro.Patinenge tichiita zviedzo, tinofanira kusarudza nzira dzakakodzera dzehuwandu maererano nemiedzo yedu pachedu.

Primer design nheyo:

Dhizaini yeprimer yeqPCR yakanangana nekuita kweamplification uye kujeka kwechigadzirwa.Naizvozvo, kugadzira zvakanaka maprimers inhanho yekutanga yekubudirira qPCR.Mukugadzira primer, zvinotevera misimboti inofanirwa kutariswa kana ikasangana nemusimboti weyakajairwa primer dhizaini:

1. Kureba kwechidimbu chinotarirwa kunodzorwa pakati pe100 ne300 bp;

2. Muchinjikwa-exon dhizaini kudzivirira kufurirwa kwegenomic DNA;

3. Iwo maprimers akagadzirwa anoda kuongororwa kuti awedzere kushanda zvakanaka, uye chete kana iyo amplification inosvika pachiyero (90-110%) inogona kushandiswa pachiyero chekuyedza;

4. Primer concentration inowanzogadziriswa pakati pe0.1uM uye 1.0uM.

Sarudzo yeROX:

Mukuita kwehuwandu hwekuita, ROX inogona kugadzirisa iyo optical nzira musiyano, pipetting kukanganisa kana vhoriyamu musiyano unokonzerwa nekubuda uye condensation zvakafanana, kuvandudza kudzokorora kwemhedzisiro.Nekudaro, zvinofanirwa kucherechedzwa kuti kusarudzwa kweROX kwakabatana nechiridzwa.Kana iyo qPCR chiridzwa chine basa rekugadzirisa otomatiki musiyano pakati pemakomba, haidi kuwedzera ROX;kana zvisina kudaro, inoda kuwedzera ROX kururamisa.Vadiki vanodyidzana mukutenga reagents vanofanirwa kuenderana nechishandiso chinoshandiswa kusarudza iyo chaiyo ROX, dzivirira gare gare kukanganisa.

Kugadzirira reaction system:

Reaction mavhoriyamu e20ul uye 50ul anosarudzwa.Zvinhu zvinotevera zvinofanirwa kutariswa kana iyo system inogadzirwa:

1. Iyo reaction system inoda kugadzirirwa nekufefetedza mu-ultra-clean workbench, ddH nyowani.₂O inoshandiswa pakuedza kwega kwega;

2. Chiyedzo chega chega chinoda kugadzirira NTC kuti ione kana pane kusvibiswa muhurongwa, uye peya peya yekutanga inoda kuita NTC pakugadzirira hurongwa;

3. Kuti uone kana pane gDNA yakasara muRNA template, NRT inogona kugadzirirwa sampuli imwe neimwe kuti ionekwe;

4. Paunenge uchigadzirira sisitimu, zvinokurudzirwa kuita kanenge 3 tekinoroji kudzokorora kune imwe sampuli;

5. Kana template iri cDNA, inokurudzirwa kunyura 5-10 nguva kuderedza inhibition effect ye reverse transcription system pakuedza kweqPCR.Zviri nani kuongorora huwandu hwetemplate ne gradient, kuitira kuti kukosha kweCT kuwire pakati pe20-30;

6. Sarudza nhamba inodiwa yemaitiro, kuwedzera ne5-10% pahwaro hwehuwandu hwekuita, uye kuverenga nhamba yehuwandu hwekugadzirisa;

7, iyo sisitimu inogadzirirwa uchishandisa iyo premix musimboti, kusanganisa mushure me centrifugation uye kuona kuti hapana mabhuru;

8, Sezvinobvira pakusarudza kutsigira zvinodyiwa.

Inoenderana RT-qPCR Kit