Teko ea RT-qPCR e kenyelletsa tlhahiso ea RNA le tlhahlobo ea boleng, phetisetso ea morao-rao le mehato e meraro ea qPCR, mohato ka mong o na le litemoso tse ngata, re tla li hlahisa ka botlalo ka tlase.

Ⅰ.tlhahlobo ea boleng ba RNA

Tekong ea RT-qPCR, ka mor'a ho phethoa ha ho ntšoa ha RNA, boleng ba RNA bo hloka ho hlahlojoa, 'me teko ea ho latela e ka etsoa feela ka mor'a hore e be le mangolo.Mekhoa ea ho hlahloba e kenyelletsa spectrophotometer, Agilent gel electrophoresis, Agilent 2100 analysis, eo har'a eona e sebelisoang ka ho fetisisa spectrophotometer le agarose gel electrophoresis mokhoa oa ho lemoha.Ho lokela ho hlokomeloa hore mekhoa ena e 'meli e lokela ho sebelisoa hammoho ho phethela ho lemoha le ho hlahloba mahloriso a RNA, bohloeki le botšepehi, e le ho netefatsa boleng ba RNA.

Related RNA Isolation Kit: 

Cell Total RNA Isolation Kit

RNA e hloekisitsoeng haholo le ea boleng bo holimo e ka fumanoa liseleng tse fapaneng tsa setso ka 11min.

Animal Total RNA Isolation Kit

Ka potlako le ka mokhoa o atlehileng o ntša RNA ea boleng bo holimo le ea boleng bo holimo ho tsoa liseleng tse fapaneng tsa liphoofolo.

Spectrophotometer:

The spectrophotometer e sebelisoa haholo-holo ho fumana hore na RNA e hokae le ho hloeka, empa ha e khone ho lemoha botšepehi ba RNA le masala a genomic.Har'a tsona, A260/280 le A260/230 ke litekanyo tsa bohlokoa tsa ho lemoha bohloeki ba RNA, 'me bohloeki ba RNA bo ka bonoa ho latela ho feto-fetoha ha boleng ba bona:

1. 1.9< A260/280< 2.1, e bontšang hore bohloeki ba RNA bo molemo;A260/280<1.9, e bontšang hore ho ka ’na ha e-ba le masala a protheine ho RNA;A260/280> 2.1, e bontšang ho senyeha ha karolo ea RNA ho ka bang teng, e ka tiisoang ka ho eketsehileng ke agarose gel electrophoresis.

2. 2.0< A260/230< 2.2, e bontšang hore bohloeki ba RNA bo molemo;A260/230< 2.0, e bontšang hore ho ka ’na ha e-ba le mesaletsa ea li-reagents tse phelang ho RNA, tse kang phenols, ethanol kapa tsoekere.

Agarose gel electrophoresis:

Agarose gel electrophoresis assay e ka hlahloba botšepehi ba RNA, liphatsa tsa lefutso le liprotheine, empa ha e khone ho lekanya ka nepo bongata ba RNA kapa ho lemoha mesaletsa ea li-organic reagents.Nka litempele tsa eukaryotic RNA mohlala:

1. RNA e ne e le tlas'a agarose gel electrophoresis.Haeba ho ne ho e-na le lihlopha tse tharo feela tsa 28sRNA, 18sRNA le 5.8sRNA 'mapeng oa gel, e bontša hore RNA e ntšitsoeng e ntse e tiile.Haeba ho na le ketsahalo e hulang, e bontša ho senyeha ha karolo ea RNA.

2. Haeba ho na le sehlopha se le seng se khanyang pakeng tsa lesoba la sekhomaretsi le sehlopha sa 28sRNA, ho ka 'na ha e-ba le masalla a DNA a genomic.

3. Haeba lihlopha li hlaha ka sekoting sa sekhomaretsi, se bontša hore ho ka 'na ha e-ba le masala a protheine le lintho tse ling tsa macromolecular.

Ⅱ. Phetolelo ea morao

Kamora hore tlhahiso ea RNA e phetheloe, e hloka ho khutlisetsoa cDNA bakeng sa liteko tse latelang, kahoo mohato oa ho khutlisa o bohlokoa.Reverse transcript e tla hlahisoa ho tsoa khethong ea reverse transcriptase le primer:

Phetola khetho ea transcriptase:

Lingoloa tse tloaelehileng tsa reverse transcriptase li kenyelletsa AMV RTase le MMLV RTase.RNase H ea AMV RTase e na le ts'ebetso e matla, bolelele bo khuts'oane ba ho kopanya, palo e tlase ea motsoako le botsitso bo botle ba mocheso (42 ~ 55 ℃).Ts'ebetso ea RNase H ea MMLV RTase e fokola, bolelele ba ho kopanya bo bolelele, palo ea motsoako e phahame, 'me botsitso ba mocheso bo bobe (37 ~ 42 ℃).

Hobane enzyme ea RNase H e na le ts'ebetso ea thempleite ea RNA e nyenyefatsang, MMLV e nang le ts'ebetso e fokolang ea RNase H e lokela ho khethoa ka mokhoa o ikhethileng nakong ea ho ngoloa ka morao, 'me ka mor'a boenjiniere ba morao-rao ba liphatsa tsa lefutso, botsitso ba mocheso oa MMLV bo fihlile tekanyo ea boleng.Ho nka ForegeneForeasy Reverse Transcriptase(M-MLV for reverse transcript) e le mohlala, ke reverse transcriptase e ncha e hlahisoang ke libaktheria tsa E. coli tse sebelisang theknoloji ea ho kopanya liphatsa tsa lefutso.Ke DNA polymerase e kopantsoeng hape e kopanyang DNA e tlatsetsang ho tloha ho RNA, DNA, kapa RNA: DNA ea mofuta o le mong.Ha e na ts'ebetso ea RNase H, botsitso bo matla, kamano e matla ea RNA, le kutloisiso e phahameng ea ho lemoha.

 

Foreasy Reverse Transcriptase(M-MLV for reverse transcript)

Khetho ea primer:

Ka kakaretso li-primers tsa RT li oela ka mekhahlelo e meraro: oligo dT, li-primers tse sa fetoheng, le li-primers tse ikhethileng tsa lefutso.Khetha li-primers tse loketseng ho sebelisoa ho latela litlhoko tse fapaneng tsa liteko.

1. Haeba template e le ea tlhaho ea eukaryotic 'me cDNA ea morao e sebelisoa bakeng sa ho hōlisa PCR ka tloaelo, Oligo (dT) e buelloa;Haeba teko e latelang e sebelisoa feela bakeng sa qPCR, Oligo (dT) e khothaletsoa hore e tsoakoe le li-primers tse sa fetoheng ho ntlafatsa ts'ebetso ea ho fetolela ka morao.

2. Haeba template e tsoa ho prokaryotes, Random Primers kapa li-primers tse khethehileng tsa lefutso li lokela ho khethoa bakeng sa phetolelo e khutlisetsang morao.

Ⅲ.qPCR

Fluorescence quantification e hlalositsoe haholo ho tsoa khethong ea mekhoa ea lipalo, melao-motheo ea moralo oa mantlha, khetho ea ROX, tlhophiso ea sistimi ea karabelo le maemo a karabelo, jj.

Khetho ea mekhoa e mengata:

Mekhoa ea lipalo e arotsoe ka mekhoa ea palo e lekanyelitsoeng le mekhoa ea palo e felletseng.Relative quantification e ka sebelisoa ho lemoha phello ea mekhoa e itseng ea phekolo ho polelo ea liphatsa tsa lefutso, ho lemoha phapang ea liphatsa tsa lefutso ka linako tse fapaneng le ho bapisa phapang ea liphatsa tsa lefutso liseleng tse fapaneng.Ka ho feletseng quantification ka lemoha palo ea nucleic acid ka kokoana-hloko joalo-joalo.Ha re etsa liteko, re tlameha ho khetha mekhoa e nepahetseng ea palo ho latela liteko tsa rona.

Melao-motheo ea mantlha ea moralo:

Moralo oa primer bakeng sa qPCR o amana ka kotloloho le bokhoni ba ho holisa le boleng ba sehlahisoa.Ka hona, ho rala ka nepo li-primers tse ntle ke mohato oa pele oa qPCR e atlehileng.Moqapi oa primer, melao-motheo e latelang e lokela ho eloa hloko ha e kopana le molao-motheo oa moralo o tloaelehileng oa primer:

1. Bolelele ba sekhechana se shebiloeng bo laoloa pakeng tsa 100 le 300 bp;

2. Moralo oa li-cross-exon ho qoba tšusumetso ea DNA ea genomic;

3. Li-primers tse entsoeng li hloka ho lekoa bakeng sa katleho ea ho hōlisa, 'me ke feela ha katleho ea amplification e fihla boemong ba (90-110%) e ka sebelisoang bakeng sa liteko tsa bongata;

4. Primer concentration hangata e ntlafatsoa lipakeng tsa 0.1uM le 1.0uM.

Khetho eaROX:

Ts'ebetsong ea karabelo ea bongata, ROX e ka fetola phapang ea tsela ea optical, phoso ea pipetting kapa phapang ea molumo e bakoang ke mouoane le condensation ka mokhoa o ts'oanang, ho ntlafatsa ho pheta-pheta ha liphetho.Leha ho le joalo, ho lokela ho hlokomeloa hore khetho ea ROX e amana le sesebelisoa.Haeba sesebelisoa sa qPCR se na le ts'ebetso ea ho lokisa phapang lipakeng tsa masoba, ha se hloke ho eketsa ROX;ho seng joalo, e hloka ho eketsa khalemelo ea ROX.Balekane ba banyenyane ha ba reka li-reagents ba tlameha ho latela sesebelisoa se sebelisoang ho khetha ROX e nepahetseng, qoba liphoso tsa morao-rao.

Ho lokisoa ha sistimi ea karabelo:

Meqolo ea karabelo ea 20ul le 50ul e khethoa.Lintlha tse latelang li lokela ho eloa hloko ha tsamaiso e etsoa:

1. Sistimi ea karabelo e hloka ho lokisoa ka moea o kenang ka har'a benche ea ho sebetsa e hloekileng haholo, ddH e ncha.₂O e sebelisoa bakeng sa teko ka 'ngoe;

2. Teko e 'ngoe le e 'ngoe e hloka ho lokisa NTC ho netefatsa hore na ho na le tšilafalo tsamaisong, 'me para e 'ngoe le e 'ngoe ea lisebelisoa e lokela ho etsa NTC ha e lokisa tsamaiso;

3. Ho bona hore na ho na le masalla a gDNA template ea RNA, NRT e ka lokisoa bakeng sa sampole ka 'ngoe bakeng sa ho fumanoa;

4. Ha u lokisetsa tsamaiso, ho kgothaletswa ho etsa bonyane ho pheta-pheta 3 ea theknoloji bakeng sa sampuli e le 'ngoe;

5. Ha thempleite e le cDNA, ho kgothaletswa ho hlapolla makgetlo a 5-10 ho fokotsa sephetho sa ho thibela reverse transcription system tekong ya qPCR.Ho molemo ho hlahloba bongata ba template ka gradient, e le hore boleng ba CT bo oela pakeng tsa 20-30;

6. Etsa qeto ea palo e hlokahalang ea liketso, eketsa ka 5-10% motheong oa palo ea liketso, 'me u bale palo ea tlhophiso ea molumo;

7, tsamaiso e lokiselitsoe ho sebelisa molao-motheo oa premix, ho kopanya ka mor'a centrifugation le ho netefatsa hore ha ho na li-bubble;

8, Ho fihlela moo ho ka khonehang ho khetha lisebelisoa tse tšehetsang.

E amanang le RT-qPCR Kit