Zvinonyatsozivikanwa kuti pakati pedzidziso, RNA ndiyo murevereri wezvinyorwa pakati peDNA uye mapuroteni kutaura.Kuenzaniswa nekuonekwa kweDNA, kuwonekwa kweRNA kunogona kuratidza zvine mutsindo mataurirwo ejene muzvinhu zvipenyu.Kuedza kunosanganisira RNA kunosanganisira: qRT-PCR, RNA-Seq, uye fusion gene kuona, etc. Kubva pane maitiro eRNA pachayo (shuga yeRNA ine rimwe boka remahara rehydroxyl kupfuura shuga yeDNA), pamwe chete nenhamba yakawanda yeRNases munharaunda, RNA inonyanya kugadzikana uye iri nyore kuderedzwa kupfuura DNA.Tsvina mukati, marara kunze, kana mhando yeRNA isina kunaka, ipapo mibairo yekuedzwa inofanira kunge isingagutsi, yakanyatso kuratidzwa se data risiri chairo kana kusadzokororwa zvakanaka.Naizvozvo, kutarisisa kwakanyanya kunofanirwa kubhadharwa pakugadziriswa kweRNA, uye chinongedzo chekutonga kwemhando yakakosha zvakare kuve nechokwadi chechokwadi uye chokwadi che data rekuyedza rinotevera.

Nekutonga kwemhando yeRNA, pane kazhinji nzira dzinotevera dzinowanzoshandiswa:

• Mifananidzo ye Spectrophotometry
• agarose gel electrophoresis
• Agilent Bioanalyzer
• chaiyo-nguva fluorescent quantitative PCR
• Qubit fluorescent dhayi nzira

01 Mifananidzo ye Spectrophotometry

RNA yakabatanidza zvisungo zvakapetwa kaviri uye ine peak yekunyura pane wavelength ye260nm.Zvinoenderana nemutemo waLambert-Beer, isu tinokwanisa kuverenga iyo RNA yekumisikidza kubva paabsorption peak pa260nm.Pamusoro pezvo, isu tinogona zvakare kuverenga kuchena kweRNA zvichienderana nereshiyo ye260nm, 280nm uye 230nm yekunyudza peaks.280nm uye 230nm ndiwo nhongonya dzemapuroteni uye mamorekuru madiki, zvichiteerana.Chiyero cheA260 / A280 uye A260 / A230 chekodzero yeRNA yakachena inofanira kuva yakakura kudarika 2. Kana iri pasi pe2, zvinoreva kuti kune mapuroteni kana kusvibiswa kwema molecule mumuenzaniso weRNA uye inoda kucheneswa zvakare.Kusvibiswa masosi kuchakanganisa kuyedza kwakadzika, sekuvharidzira kugona kwePCR maitiro, zvichikonzera mhedzisiro yehuwandu.Kuchena kweRNA kune pesvedzero huru pane dzinotevera mhedzisiro, saka spectrophotometry inowanzova yakakosha yekudzora mhando yekubatanidza munhanho yekutanga mukuyedza nucleic acid.

Mufananidzo 1. Chimiro RNA / DNA Absorption Spectrum

02 Agarose gel electrophoresis

Pamusoro pekuchena, kuvimbika kweRNA zvakare chimwe chezviratidzo zvakakosha zvekutonga kunaka kweRNA.Kusvibiswa kweRNA kunotungamira kune nhamba huru yezvimedu zvipfupi mumuenzaniso, saka nhamba yeRNA zvimedu zvinogona kunyatsoonekwa uye zvakavharwa nereferensi yereferensi ichaderedzwa.RNA kutendeseka kunogona kuongororwa ne electrophoresis yeRNA yakazara pane 1% agarose gel.Iyi nzira inogona kugadzirisa gel iwe pachako, kana kushandisa yakafanogadzirirwa E-Gel ™ Sisitimu yekuyedza kutendeseka.Inopfuura 80% yeRNA yakazara ribosomal RNA, yakawanda yacho inoumbwa ne28S uye 18S rRNA (mune mammalian systems).Hunhu hwakanaka RNA icharatidza mabhawa maviri akajeka akajeka, ayo ari 28S uye 18S mabhawa anopenya, zvichiteerana, pa5 Kb uye 2 Kb, uye reshiyo ichave iri pedyo ne2: 1.Kana iri munzvimbo yakapararira, zvinoreva kuti RNA sampuli inogona kunge yakaderedzwa, uye inokurudzirwa kushandisa nzira inotsanangurwa gare gare kuti uwedzere kuedza kunaka kweRNA.

Mufananidzo 2. Kuenzanisa kwakaderedzwa (mutsara 2) uye intact RNA (lane 3) pane agarose gel electrophoresis

03 Agilent Bioanalyzer

Mukuwedzera kune agarose gel electrophoresis nzira inotsanangurwa pamusoro apa, iyo inogona kutibatsira kuziva kuvimbika kweRNA zviri nyore uye nokukurumidza, tinogonawo kushandisa Agilent bioanalyzer kuti tione kuvimbika kweRNA.Inoshandisa musanganiswa we microfluidics, capillary electrophoresis, uye fluorescence kuongorora RNA kusungwa uye kuvimbika.Nekushandisa iyo yakavakirwa-mukati algorithm yekuongorora chimiro cheiyo RNA sampuli, iyo Agilent bioanalyzer inogona kuverenga referensi RNA kutendeseka kukosha, RNA Integrity Number (inozonzi RIN) [1].Iyo yakakura kukosha kweRIN, iyo yakakwirira kuvimbika kweRNA (1 yakanyanya kuderedzwa, gumi ndiyo yakanyanya kukwana).Zvimwe zviedzo zvinosanganisira RNA zvinokurudzira kushandisa RIN separameter yekuongorora kwemhando.Kutora zviyedzo zvekuteedzera zvakakwirira (zvino zvinozonzi NGS) semuenzaniso, nhungamiro yeOncomine ™ Human Immune Repertoire, iyo inoshandiswa kuona B cell uye T cell antigen receptors muThermo Fisher's Oncomine panel series, inoratidza kuti sampuli dzine RIN dzinokosha kupfuura 4, Kuwedzera kunobudirira kuyerwa uye mafiritsi anogona kuverengwa (Figure 3).Kune akasiyana-siyana anokurudzirwa marengeti emapaneru akasiyana, uye kazhinji RIN yepamusoro inogona kuunza yakawanda inoshanda data.

Mufananidzo 3, muOncomine™ Human Immune Repertoire kuyedza, masampuli ane RIN anopfuura mana anogona kuona kuverenga kunoshanda uye T cell clones.【2】

Nekudaro, iyo RIN kukosha zvakare ine zvimwe zvisingakwanisi.Kunyangwe RIN ine hukama hwepamusoro nemhando ye data yekuedza yeNGS, haina kukodzera sampuli dzeFFPE.Masampuli eFFPE akabatwa nemakemikari kwenguva yakareba, uye iyo RNA yakabviswa kazhinji ine yakaderera RIN kukosha.Zvisinei, izvi hazvirevi kuti data inoshanda yekuedza inofanira kunge isingafadzi.Kuti tinyatso ongorora mhando yemasampuli eFFPE, tinoda kushandisa zviyero kunze kweRIN.Pamusoro peRIN, Agilent bioanalyzer inogona zvakare kuverenga DV200 kukosha seyekuongorora parameter yeRNA mhando.DV200 iparameter inoverenga chikamu chezvidimbu zvakakura kudarika 200 bp mumuenzaniso weRNA.DV200 iri nani chiratidzo cheFFPE mhando yemhando pane RIN.Kune iyo RNA yakabviswa neFFPE, ine hukama hwakanyanya nehuwandu hwemajini anogona kunyatsoonekwa uye kusiyana kwemajini [3].Kunyangwe zvazvo DV200 ichikwanisa kugadzirisa zvinoshomeka mukuonekwa kwemhando yeFFPE, iyo Agilent bioanalyzer haisati yakwanisa kunyatsoongorora matambudziko emhando mumasampunzi eRNA, kusanganisira kana paine ma inhibitors mumasampuli.Inhibitors pachayo inogona kukanganisa kukwidziridzwa kwezviyedzo zvepasi uye kuderedza huwandu hwe data inobatsira.Kuti tizive kana paine inhibitor mumuenzaniso, tinogona kutora chaiyo-nguva fluorescent quantitative PCR nzira inotsanangurwa inotevera.

04 chaiyo-nguva fluorescent quantitative PCR

Iyo chaiyo-nguva fluorescent quantitative PCR nzira haigone kungoona iyo inhibitors mumuenzaniso, asi zvakare inonyatso ratidza kunaka kweRNA mune yeFFPE sampu.Zvichienzaniswa neAgilent biological analyzers, real-time fluorescence quantitative zviridzwa zvinonyanya kufarirwa mumarabhoritari makuru ezvipenyu nekuda kwekushandisa kwavo kwakafara.Kuti tiedze kunaka kwemasamples eRNA, tinongoda kutenga kana kugadzirira maprimer probes emagetsi emukati, akadai seGUSB (Cat no. Hs00939627).Nekushandisa iyi seti yemaprimers, probes uye zviyero (yakazara RNA yekuzivikanwa kusinganzwisisike) kuita yakakwana yehuwandu kuyedza, iyo inoshanda RNA fragment concentration inogona kuverengerwa seyero yekuongorora yeRNA mhando (Functional RNA Quantitation (FRQ) kwenguva pfupi).Mukuyedzwa kweNGS, takaona kuti iyo FRQ yemasampuli eRNA ine hukama hwakanyanya nehuwandu hwe data hunoshanda.Kune ese masampuli makuru kupfuura 0.2ng/uL FRQ, ingangoita 70% yekuverenga inogona kunyatsovhara kutevedzana kwereferenzi (Mufananidzo 4).

Mufananidzo 4, kukosha kweFRQ kunowanikwa nefluorescence quantitative nzira ine kuwirirana kwakanyanya (R2> 0.9) nemashoko anobudirira akawanikwa mukuedza kweNGS.Mutsara mutsvuku ndiyo FRQ kukosha yakaenzana ne0.2 ng/uL (log10 = -0.7).【4】

Pamusoro pekushanda kune FFPE samples, iyo chaiyo-nguva yehuwandu PCR nzira inogona zvakare kunyatso tarisa inhibitors mumasampuli.Tinogona kuwedzera sampuli kuti ionekwe mukuita system neInternal Positive Control (IPC) uye Assay yayo, uye tozoita fluorescence quantification kuti uwane kukosha kweCt.Kana iyo Ct kukosha kwasara kumashure kweiyo Ct kukosha mune isina-sample reaction, inoratidza kuti iyo inhibitor iripo mumuenzaniso uye inhibitsa kukwidziridzwa kwakanaka mukuita.

05 Qubit fluorescent dhayi nzira

Qubit Fluorometer ndiyo inonyanya kushandiswa diki mudziyo weiyo nucleic acid yekumisikidza uye kuona kuchena, iri nyore kushanda uye inowanikwa mune ingangoita yega molecular biology rabhoritari.Iyo inonyatso kuverenga kuunganidzwa kweiyo nucleic acid nekuona uye nucleic acid-inosunga fluorescent dhayi (Qubit yekuona reagent).Qubit ine yakanyanya kunzwisiswa uye yakasarudzika, uye inokwanisa kunyatso kuverengera RNA pasi kusvika pg/µL concentration.Pamusoro peiyo inosanozivikanwa kugona kuverengera nenzira kwayo nucleic acid concentration, Thermo Fisher yazvino modhi itsva, Qubit 4.0, inogona zvakare kuona kuvimbika kweRNA.Qubit 4.0's RNA yekuongorora system (RNA IQ Assay) inoona kuvimbika kweRNA nekuona panguva imwe chete madhayi maviri efluorescent.Aya madhayi maviri efluorescent anogona kusunga kune zvimedu zvakakura uye zvidimbu zvidiki zveRNA, zvichiteerana.Aya madhayi maviri efluorescent anoratidza chikamu chezvidimbu zvakakura zveRNA mumuenzaniso, uye kubva pane iyi IQ (Kutendeseka uye Unhu) kukosha kunomiririra RNA unhu hunogona kuverengwa.Iko kukosha kweIQ kunoshanda kune ese ari maviri FFPE uye asiri-FFPE samples, uye ine simba guru pane inotevera kutevedzana kwemhando.Tichitora zviedzo zveNGS semuenzaniso, muyedzo yeRNA-Seq bvunzo yakaitwa paIon torrent ™ papuratifomu, masampuli mazhinji ane maIQ makuru kupfuura mana aive neinenge 50% kuverenga kwakanaka (Mufananidzo 5).Kuenzaniswa nemaitiro ataurwa pamusoro apa, Qubit IQ Assay haisi nyore nyore kushanda uye inotora nguva shoma (mukati memaminitsi mashanu), asiwo ine kuwirirana kukuru pakati peyero yakayerwa parameter IQ kukosha uye data data yezviyedzo zvepasi.

Mufananidzo 5, pane kuwirirana kukuru pakati peQubit RNA IQ kukosha uye mamepu anoverengwa eRNA-Seq.【5】

Kuburikidza nesumo iri pamusoro, ndinotenda kuti munhu wese ane nzwisiso yakakwana yemhando dzakasiyana dzeRNA nzira dzekutonga.Mukuita, unogona kusarudzanzira inoenderana maererano nemhando yemuenzaniso uye zviridzwa zviripo.Chete nekudzora mhando yeRNA zvakanaka tinogona kudzivirira kutadza kwezviedzo zvinotevera zvinokonzerwa nehurombo hwemhando yemhando, nekudaro tichichengetedza nguva yakakosha, simba uye mutengo.

Reference products:

Mhuka Yese RNA Isolation Kit

Sero Yese RNA Isolation Kit

mareferensi

【1】Schroeder, A., Mueller, O., Stocker, S. et al.Iyo RIN: nhamba yeruvimbiko yeRNA yekugovera hunhu hwekuvimbika kune zviyero zveRNA.BMC Molecular Biol 7, 3 (2006).https:// doi .org/10.1186/1471-21 99-7-3

【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, Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples, Toxicological Sciences, Volume 3, 3 Nyamavhuvhu 1720, Issue 1720https://doi.org/10.1093/toxsci/