Nws yog paub zoo tias nyob rau hauv lub hauv paus dogma, RNA yog transcriptional mediator ntawm DNA thiab protein qhia.Piv nrog rau kev tshawb pom ntawm DNA, kev tshawb pom ntawm RNA tuaj yeem cuam tshuam ntau dua qhov pom ntawm cov noob hauv cov kab mob.Cov kev sim uas muaj RNA suav nrog: qRT-PCR, RNA-Seq, thiab fusion gene detection, thiab lwm yam. Raws li cov yam ntxwv ntawm RNA nws tus kheej (lub nplhaib qab zib ntawm RNA muaj ib pab pawg dawb hydroxyl ntau dua li cov suab thaj ntawm DNA), ua ke nrog ntau tus RNases hauv ib puag ncig, RNA tsis ruaj khov thiab yooj yim rau degraded dua DNA.Cov khib nyiab hauv, khib nyiab tawm, yog tias qhov zoo ntawm RNA tsis zoo, tom qab ntawd cov txiaj ntsig kev sim yuav tsum tsis txaus siab, tshwj xeeb tshaj yog cov ntaub ntawv tsis raug lossis tsis zoo rov ua dua.Yog li ntawd, yuav tsum tau saib xyuas ntau dua rau kev ua haujlwm ntawm RNA, thiab kev sib txuas ntawm kev tswj xyuas zoo kuj tseem ceeb dua los xyuas kom meej qhov tseeb thiab qhov tseeb ntawm cov ntaub ntawv sim tom ntej.

Rau kev tswj kom zoo ntawm RNA, feem ntau muaj cov hauv qab no uas nquag siv:

• Spectrophotometric
• Electrophoresis agarose gel yog siv
• Agilent Bioanalyzer
• fluorescent quantitative PCR lub sij hawm
• Qubit fluorescent dye method

01 Spectrophotometer

RNA tau sib txuas ob daim ntawv cog lus thiab muaj qhov nqus siab kawg ntawm lub wavelength ntawm 260nm.Raws li Lambert-Beer txoj cai, peb tuaj yeem xam RNA concentration los ntawm qhov siab tshaj plaws ntawm 260nm.Tsis tas li ntawd, peb tuaj yeem xam qhov purity ntawm RNA raws li qhov sib piv ntawm 260nm, 280nm thiab 230nm absorption peaks.280nm thiab 230nm yog qhov nqus siab tshaj plaws ntawm cov protein thiab cov molecules me, feem.Qhov piv ntawm A260 / A280 thiab A260 / A230 ntawm qhov tsim nyog RNA purity yuav tsum ntau dua 2. Yog tias nws tsawg dua 2, nws txhais tau hais tias muaj cov protein lossis cov kab mob me me hauv cov qauv RNA thiab yuav tsum tau ua kom huv dua.Cov chaw sib kis yuav cuam tshuam rau cov kev sim qis qis, xws li inhibiting qhov ua kom zoo dua ntawm PCR cov tshuaj tiv thaiv, ua rau cov txiaj ntsig tsis raug.Lub purity ntawm RNA muaj kev cuam tshuam zoo rau cov txiaj ntsig tom qab, yog li spectrophotometric feem ntau yog qhov tseem ceeb ntawm kev tswj xyuas qhov txuas hauv thawj kauj ruam hauv kev sim nucleic acid.

Daim duab 1. Hom RNA/DNA Absorption Spectrum

02 Agarose gel electrophoresis

Ntxiv nrog rau kev ua kom huv huv, kev ncaj ncees ntawm RNA kuj yog ib qho tseem ceeb rau kev txiav txim siab zoo ntawm RNA.Qhov degradation ntawm RNA yuav ua rau muaj ntau qhov luv luv hauv cov qauv, yog li tus naj npawb ntawm RNA fragments uas tuaj yeem kuaj pom tau zoo thiab npog los ntawm cov ntaub ntawv siv tau yuav raug txo.RNA kev ncaj ncees tuaj yeem tshawb xyuas los ntawm electrophoresis ntawm tag nrho RNA ntawm 1% agarose gel.Txoj kev no tuaj yeem teeb tsa lub gel koj tus kheej, lossis siv prefabricated E-Gel ™ System rau kev kuaj kev ncaj ncees.Ntau tshaj 80% ntawm tag nrho RNA yog ribosomal RNA, feem ntau yog tsim los ntawm 28S thiab 18S rRNA (hauv cov tsiaj txhu).Qhov zoo RNA yuav pom ob lub teeb ci ntsa iab, uas yog 28S thiab 18S ci ntsa iab, feem, ntawm 5 Kb thiab 2 Kb, thiab qhov sib piv yuav zoo li ze rau 2: 1.Yog tias nws nyob hauv lub xeev diffuse, nws txhais tau hais tias tus qauv RNA yuav raug degraded, thiab nws raug nquahu kom siv cov txheej txheem piav qhia tom qab txhawm rau ntsuas qhov zoo ntawm RNA.

Daim duab 2. Kev sib piv ntawm degraded (txoj kab 2) thiab zoo RNA (txoj kab 3) ntawm agarose gel electrophoresis

03 Agilent Bioanalyzer

Ntxiv rau agarose gel electrophoresis txoj kev tau piav qhia saum toj no, uas tuaj yeem pab peb txheeb xyuas qhov kev ncaj ncees ntawm RNA yooj yim thiab nrawm, peb kuj tuaj yeem siv Agilent bioanalyzer los txiav txim siab txog kev ncaj ncees ntawm RNA.Nws siv kev sib xyaw ntawm microfluidics, capillary electrophoresis, thiab fluorescence los ntsuas RNA concentration thiab kev ncaj ncees.Los ntawm kev siv cov txheej txheem built-in los txheeb xyuas qhov profile ntawm tus qauv RNA, Agilent bioanalyzer tuaj yeem xam tus nqi RNA kev ncaj ncees, RNA Integrity Number (tom qab no hu ua RIN) [1].Qhov loj dua tus nqi ntawm RIN, qhov siab dua ntawm RNA (1 yog degraded heev, 10 yog qhov ua tiav tshaj plaws).Qee qhov kev sim nrog RNA qhia siv RIN ua qhov ntsuas rau kev ntsuas zoo.Ua cov kev sim siab dhau los ua ntu zus (tom qab no hu ua NGS) ua piv txwv, cov lus qhia ntawm Oncomine™ Human Immune Repertoire, uas yog siv los txheeb xyuas B cell thiab T cell antigen receptors hauv Thermo Fisher's Oncomine panel series, qhia tias cov qauv nrog RIN muaj nuj nqis ntau dua 4, Kev ntsuas tau zoo dua thiab nyeem tau ntau dua 3 clones).Muaj ntau qhov kev pom zoo sib txawv rau cov vaj huam sib luag sib txawv, thiab feem ntau RIN siab dua tuaj yeem nqa cov ntaub ntawv zoo dua.

Daim duab 3, nyob rau hauv Oncomine™ Human Immune Repertoire thwmsim, cov qauv nrog RIN ntau dua 4 tuaj yeem kuaj pom kev nyeem tau zoo dua thiab T cell clones.【2】

Txawm li cas los xij, tus nqi RIN kuj muaj qee qhov kev txwv.Txawm hais tias RIN muaj kev sib raug zoo nrog qhov zoo ntawm NGS cov ntaub ntawv sim, nws tsis haum rau FFPE cov qauv.FFPE cov qauv tau raug kho tshuaj rau lub sijhawm ntev, thiab cov RNA rho tawm feem ntau muaj tus nqi RIN tsawg.Txawm li cas los xij, qhov no tsis tau txhais hais tias cov ntaub ntawv zoo ntawm kev sim yuav tsum tsis txaus siab.Txhawm rau ntsuas qhov zoo ntawm FFPE cov qauv, peb yuav tsum siv cov kev ntsuas uas tsis yog RIN.Ntxiv rau RIN, Agilent bioanalyzer tseem tuaj yeem suav DV200 tus nqi raws li qhov ntsuas ntsuas ntawm RNA zoo.DV200 yog ib qho parameter uas xam qhov feem ntawm cov fragments loj dua 200 bp hauv ib qho qauv RNA.DV200 yog qhov qhia tau zoo ntawm FFPE qauv zoo dua li RIN.Rau RNA rho tawm los ntawm FFPE, nws muaj kev sib raug zoo heev nrog cov noob caj noob ces uas tuaj yeem kuaj pom tau zoo thiab muaj ntau haiv neeg [3].Txawm hais tias DV200 tuaj yeem ua rau qhov tsis zoo hauv kev tshawb pom zoo ntawm FFPE, Agilent bioanalyzer tseem tsis tuaj yeem txheeb xyuas cov teeb meem zoo hauv RNA cov qauv, suav nrog seb puas muaj cov tshuaj tiv thaiv hauv cov qauv.Inhibitors lawv tus kheej tuaj yeem cuam tshuam rau kev nthuav dav ntawm cov kev sim hauv qab thiab txo cov ntaub ntawv muaj txiaj ntsig.Txhawm rau paub seb puas muaj cov tshuaj tiv thaiv hauv cov qauv, peb tuaj yeem siv lub sijhawm tiag tiag fluorescent quantitative PCR txoj kev piav qhia tom ntej.

04 fluorescent quantitative PCR lub sij hawm

Lub sijhawm tiag tiag fluorescent quantitative PCR txoj kev tuaj yeem tsis tsuas yog kuaj xyuas cov inhibitors hauv cov qauv, tab sis kuj tseem cuam tshuam qhov zoo ntawm RNA hauv FFPE qauv.Piv nrog Agilent biological analyzers, cov cuab yeej siv hluav taws xob fluorescence tiag tiag tau nrov dua hauv cov chaw sim tshuaj lom neeg loj vim lawv siv dav dua.Txhawm rau kuaj qhov zoo ntawm cov qauv RNA, peb tsuas yog yuav tsum tau yuav lossis npaj cov tshuaj tiv thaiv primer rau cov noob siv sab hauv, xws li GUSB (Cat no. Hs00939627).Los ntawm kev siv cov txheej txheem ntawm primers, sojntsuam thiab cov qauv (tag nrho RNA ntawm kev paub concentration) los ua qhov kev sim ntau npaum li cas, qhov ua tau zoo ntawm RNA fragment concentration yuav raug xam raws li tus qauv ntsuas ntawm RNA zoo (Functional RNA Quantitation (FRQ) luv luv).Hauv kev xeem NGS, peb pom tias FRQ ntawm RNA cov qauv muaj kev sib raug zoo nrog cov ntaub ntawv zoo.Rau tag nrho cov qauv ntau dua 0.2ng / uL FRQ, tsawg kawg yog 70% ntawm cov ntawv nyeem tuaj yeem npog cov ntaub ntawv siv tau zoo (Daim duab 4).

Daim duab 4, FRQ tus nqi kuaj pom los ntawm fluorescence quantitative txoj kev muaj kev sib raug zoo heev (R2> 0.9) nrog cov ntaub ntawv zoo tau txais hauv NGS kev sim.Cov kab liab yog FRQ tus nqi sib npaug rau 0.2 ng / uL (log10 = -0.7).【4】

Ntxiv rau qhov muaj feem xyuam rau FFPE cov qauv, cov qauv PCR ntawm lub sijhawm tiag tiag kuj tuaj yeem saib xyuas cov tshuaj tiv thaiv hauv cov qauv.Peb tuaj yeem ntxiv cov qauv kom raug kuaj pom hauv cov tshuaj tiv thaiv nrog Internal Positive Control (IPC) thiab nws qhov kev soj ntsuam, thiab tom qab ntawd ua cov fluorescence kom tau txais tus nqi Ct.Yog tias tus nqi Ct poob qis tom qab tus nqi Ct hauv qhov tsis muaj qauv tshuaj tiv thaiv, nws qhia tau hais tias tus inhibitor muaj nyob rau hauv cov qauv thiab inhibits lub amplification efficiency nyob rau hauv cov tshuaj tiv thaiv.

05 Qubit fluorescent dye method

Qubit Fluorometer yog cov cuab yeej siv me me tshaj plaws rau nucleic acid concentration thiab purity nrhiav kom tau, uas yooj yim rau kev khiav lag luam thiab muaj nyob hauv yuav luag txhua qhov chaw kuaj mob molecular.Nws raug xam qhov concentration ntawm nucleic acid los ntawm kev kuaj thiab nucleic acid-binding fluorescent dye (Qubit detection reagent).Qubit muaj qhov rhiab heev thiab qhov tshwj xeeb, thiab tuaj yeem txheeb xyuas RNA kom raug mus rau pg / µL concentration.Ntxiv nrog rau qhov muaj peev xwm paub txog qhov tseeb kom muaj nuj nqis nucleic acid concentration, Thermo Fisher tus qauv tshiab tshiab, Qubit 4.0, kuj tuaj yeem kuaj xyuas qhov ncaj ncees ntawm RNA.Qubit 4.0′s RNA detection system (RNA IQ Assay) kuaj xyuas qhov ncaj ncees ntawm RNA los ntawm ib txhij kuaj pom ob qhov tshwj xeeb fluorescent dyes.Ob lub fluorescent dyes tuaj yeem khi rau cov khoom loj thiab cov khoom me me ntawm RNA, feem.Ob lub fluorescent dyes no qhia txog qhov feem ntawm cov khoom loj ntawm RNA hauv cov qauv, thiab los ntawm qhov no IQ (Kev Ncaj Ncees thiab Zoo) tus nqi sawv cev rau RNA zoo tuaj yeem suav tau.Tus nqi IQ muaj feem xyuam rau ob qho tib si FFPE thiab tsis yog-FFPE cov qauv, thiab muaj kev cuam tshuam zoo rau kev ua kom zoo dua qub.Noj NGS cov kev sim ua piv txwv, hauv RNA-Seq kev sim kev sim ua rau ntawm Ion torrent ™ platform, feem ntau cov qauv nrog IQ qhov tseem ceeb tshaj 4 muaj tsawg kawg yog 50% nyeem tau zoo (Daim duab 5).Piv nrog rau cov kev tshawb pom saum toj no, Qubit IQ Assay tsis tsuas yog yooj yim dua rau kev khiav lag luam thiab siv sijhawm tsawg dua (hauv tsib feeb), tab sis kuj muaj kev sib raug zoo ntawm qhov ntsuas ntsuas ntsuas IQ tus nqi thiab cov ntaub ntawv zoo ntawm cov kev sim hauv qab.

Daim duab 5, muaj kev sib raug zoo ntawm Qubit RNA IQ tus nqi thiab daim ntawv qhia nyeem ntawm RNA-Seq.【5】

Los ntawm cov lus qhia saum toj no, kuv ntseeg tias txhua tus neeg muaj kev nkag siab txaus ntawm RNA txoj kev tswj xyuas zoo.Hauv kev xyaum, koj tuaj yeem xaivtxoj kev sib raug raws li cov qauv qauv thiab cov cuab yeej uas twb muaj lawm.Tsuas yog los ntawm kev tswj hwm qhov zoo ntawm RNA zoo tuaj yeem peb zam qhov tsis ua tiav ntawm cov kev sim tom ntej los ntawm cov qauv tsis zoo, yog li txuag lub sijhawm muaj txiaj ntsig, lub zog thiab tus nqi.

Cov khoom siv:

Tsiaj Tag Nrho RNA Isolation Kit

Cell Total RNA Isolation Kit

cov ntaub ntawv

【1】 Schroeder, A., Mueller, O., Stocker, S. et al.RIN: tus lej RNA kev ncaj ncees rau kev muab cov txiaj ntsig kev ncaj ncees rau RNA ntsuas.BMC Molecular Biol 7, 3 (2006).https:// doi .org/10.1186/1471-21 99-7-3 ib

【2】Oncomine Human Immune Repertoire User Guide (Pub. No. MAN0017438 Rev. C.0).

【3】 Leah C Wehmas, Charles E Wood, Brian N Chorley, Carole L Yauk, Gail M Nelson, Susan D Hester, Txhim Kho Kev Ntsuas Zoo rau Kev Ntsuas RNA Tau Txais Los Ntawm Archival Formalin-Fixed Paraffin-Embedded Tissue Samples, Toxicological Sciences, Volume 7, 2015, Lub Yim Hli 20, 2017, Nplooj 27, 2019 3,https://doi.org/10.1093/toxsci/