Ndizodziwika bwino kuti pakati pa chiphunzitso chapakati, RNA ndiye mkhalapakati wolembera pakati pa DNA ndi mawu a protein.Poyerekeza ndi kuzindikira kwa DNA, kuzindikirika kwa RNA kumatha kuwonetsa bwino lomwe mawonekedwe a jini m'zamoyo.Zoyesera zokhudzana ndi RNA zikuphatikizapo: qRT-PCR, RNA-Seq, ndi kufufuza jini ya fusion, etc. Malingana ndi makhalidwe a RNA yokha (mphete ya shuga ya RNA ili ndi gulu limodzi laulere la hydroxyl kuposa mphete ya shuga ya DNA), kuphatikizapo chiwerengero chachikulu cha RNases m'chilengedwe, RNA imakhala yosakhazikika komanso yosavuta kuti iwonongeke kuposa DNA.Zinyalala mu, zinyalala kunja, ngati khalidwe la RNA si zabwino, ndiye zotsatira experimental ayenera kukhala zosakhutiritsa, makamaka kuwonetseredwa ngati deta yolakwika kapena osauka repeatability.Chifukwa chake, chidwi chochulukirapo chiyenera kuperekedwa pakukonza kwa RNA, ndipo ulalo waulamuliro wabwino ndi wofunikira kwambiri kuti zitsimikizire kulondola komanso kulondola kwa data yoyeserera.

Pakuwongolera kwabwino kwa RNA, nthawi zambiri pamakhala njira zotsatirazi:

• Spectrophotometry
• agarose gel electrophoresis
• Agilent Bioanalyzer
• zenizeni nthawi fulorosenti kuchuluka kwa PCR
• Njira yopangira utoto wa fulorosenti

01 Spectrophotometry

RNA yalumikiza zomangira zapawiri ndipo ili ndi chiwongolero cha mayamwidwe pamtunda wa 260nm.Malinga ndi lamulo la Lambert-Beer, titha kuwerengera kuchuluka kwa RNA kuchokera pachimake pamayamwidwe pa 260nm.Kuphatikiza apo, titha kuwerengeranso chiyero cha RNA molingana ndi nsonga zamayamwidwe a 260nm, 280nm ndi 230nm.280nm ndi 230nm ndi nsonga za mayamwidwe a mapuloteni ndi mamolekyu ang'onoang'ono, motsatana.Chiŵerengero cha A260 / A280 ndi A260 / A230 cha chiyero choyenerera cha RNA chiyenera kukhala chachikulu kuposa 2. Ngati sichiposa 2, zikutanthauza kuti pali mapuloteni kapena kuipitsidwa kwa mamolekyu ang'onoang'ono mu chitsanzo cha RNA ndipo chiyenera kuyeretsedwanso.Zomwe zimayipitsidwa zidzakhudza kuyesa kwapansi, monga kuletsa kukulitsa kwa machitidwe a PCR, zomwe zimapangitsa kuti pakhale zotsatira zolakwika.Kuyera kwa RNA kumakhudza kwambiri zotsatira zotsatila, kotero kuti spectrophotometry nthawi zambiri imakhala ulalo wofunikira kwambiri pakuwongolera koyambira pamayeso a nucleic acid.

Chithunzi 1. Chitsanzo cha RNA / DNA Absorption Spectrum

02 Agarose gel electrophoresis

Kuphatikiza pa chiyero, umphumphu wa RNA ndi chimodzi mwa zizindikiro zofunika poweruza khalidwe la RNA.Kuwonongeka kwa RNA kudzatsogolera ku chiwerengero chachikulu cha tizidutswa tating'ono mu chitsanzo, kotero kuti chiwerengero cha zidutswa za RNA zomwe zingathe kuzindikiridwa bwino ndikuphimbidwa ndi ndondomeko yowonetsera zidzachepetsedwa.Umphumphu wa RNA ukhoza kufufuzidwa ndi electrophoresis ya RNA yonse pa 1% gel agarose.Njirayi imatha kukonza gel nokha, kapena kugwiritsa ntchito E-Gel™ System yoyeserera poyesa kukhulupirika.Kuposa 80% ya RNA yonse ndi ribosomal RNA, yambiri yomwe ili ndi 28S ndi 18S rRNA (mu machitidwe a mammalian).Ubwino wa RNA uwonetsa mipiringidzo iwiri yowoneka bwino, yomwe ili 28S ndi 18S mipiringidzo yowala, motsatana, pa 5 Kb ndi 2 Kb, ndipo chiŵerengerocho chidzakhala pafupi ndi 2: 1.Ngati ili mu chikhalidwe chofalikira, zikutanthauza kuti chitsanzo cha RNA chikhoza kukhala chodetsedwa, ndipo tikulimbikitsidwa kugwiritsa ntchito njira yomwe tafotokoza pambuyo pake kuti mupitirize kuyesa khalidwe la RNA.

Chithunzi 2. Kufananiza zowonongeka (njira 2) ndi RNA yosasunthika (njira 3) pa agarose gel electrophoresis

03 Agilent Bioanalyzer

Kuwonjezera pa njira ya agarose gel electrophoresis yomwe yafotokozedwa pamwambapa, yomwe ingatithandize kuzindikira kukhulupirika kwa RNA mosavuta komanso mofulumira, tingagwiritsenso ntchito Agilent bioanalyzer kuti tidziwe kukhulupirika kwa RNA.Amagwiritsa ntchito kuphatikiza kwa microfluidics, capillary electrophoresis, ndi fluorescence kuti ayese ndende ya RNA ndi kukhulupirika.Pogwiritsa ntchito ma algorithm omangidwa kuti asanthule mbiri ya chitsanzo cha RNA, Agilent bioanalyzer imatha kuwerengera mtengo waumphumphu wa RNA, Nambala Yokhulupirika ya RNA (yotchedwa RIN) [1].Mtengo wokulirapo wa RIN, ndiye kuti umphumphu wa RNA umakwera kwambiri (1 ndiyonyozeka kwambiri, 10 ndiyokwanira kwambiri).Zoyeserera zina zokhudzana ndi RNA zikuwonetsa kugwiritsa ntchito RIN ngati gawo lowunika bwino.Kutenga zoyeserera zotsatizana kwambiri (zotchedwa NGS) mwachitsanzo, malangizo a Oncomine ™ Human Immune Repertoire, omwe amagwiritsidwa ntchito pozindikira B cell ndi T cell antigen receptors mu Thermo Fisher's Oncomine gulu la gulu, akuwonetsa kuti zitsanzo zokhala ndi RIN mikhalidwe yayikulu kuposa 4, Kuwerenga kogwira mtima kwambiri ndi ma clones amatha kuwerengedwa (Figure 3).Pali mitundu yosiyanasiyana yovomerezeka ya mapanelo osiyanasiyana, ndipo nthawi zambiri RIN yapamwamba imatha kubweretsa zambiri zothandiza.

Chithunzi 3, mu kuyesa kwa Oncomine ™ Human Immune Repertoire, zitsanzo zokhala ndi RIN zazikulu kuposa 4 zimatha kuzindikira zowerengera bwino komanso ma cell a T cell.【2】

Komabe, mtengo wa RIN ulinso ndi malire.Ngakhale RIN ili ndi kulumikizana kwakukulu ndi mtundu wa data yoyesera ya NGS, siyoyenera zitsanzo za FFPE.Zitsanzo za FFPE zakhala zikugwiritsidwa ntchito ndi mankhwala kwa nthawi yayitali, ndipo RNA yotengedwa nthawi zambiri imakhala ndi mtengo wochepa wa RIN.Komabe, izi sizikutanthauza kuti deta yogwira ntchito yoyesera iyenera kukhala yosakhutiritsa.Kuti tiwone bwinobwino mtundu wa zitsanzo za FFPE, tiyenera kugwiritsa ntchito miyeso ina osati RIN.Kuphatikiza pa RIN, Agilent bioanalyzer imathanso kuwerengera mtengo wa DV200 ngati gawo lowunika la mtundu wa RNA.DV200 ndi chizindikiro chomwe chimawerengera kuchuluka kwa zidutswa zazikulu kuposa 200 bp mu RNA.DV200 ndi chizindikiro chabwino cha mtundu wa FFPE kuposa RIN.Kwa RNA yotengedwa ndi FFPE, ili ndi kulumikizana kwakukulu ndi kuchuluka kwa majini omwe amatha kuzindikirika bwino komanso kusiyanasiyana kwa majini [3].Ngakhale kuti DV200 imatha kupanga zoperewera pakuzindikirika kwamtundu wa FFPE, Agilent bioanalyzer siyingathe kusanthula mwatsatanetsatane zovuta zamtundu wa RNA, kuphatikiza ngati pali zoletsa m'masampulo.Ma inhibitors okha amatha kukhudza kukulitsa luso la zoyeserera zakumunsi ndikuchepetsa kuchuluka kwa deta yothandiza.Kuti tidziwe ngati pali choletsa pachitsanzocho, titha kugwiritsa ntchito njira yeniyeni ya PCR ya fulorosenti yomwe yafotokozedwa motsatira.

04 zenizeni nthawi fulorosenti kuchuluka kwa PCR

Njira yeniyeni ya fulorosenti yochulukirachulukira ya PCR sikungozindikira zoletsa zomwe zili mu zitsanzo, komanso zimawonetsa bwino mtundu wa RNA mu zitsanzo za FFPE.Poyerekeza ndi Agilent biological analyzers, zida zenizeni zenizeni za fluorescence quantitative ndizodziwika kwambiri m'ma laboratories akuluakulu azachilengedwe chifukwa chakugwiritsa ntchito kwawo mokulirapo.Kuti tiyese mtundu wa zitsanzo za RNA, timangofunika kugula kapena kukonzekera ma probes amtundu wamtundu wamkati, monga GUSB (Cat no. Hs00939627).Pogwiritsa ntchito ma primers, probes ndi miyezo (chiwerengero cha RNA chodziwika bwino) kuti ayese kuyesa kokwanira, chigawo chogwira ntchito cha RNA fragment chikhoza kuwerengedwa ngati muyeso wowunika wa khalidwe la RNA (Functional RNA Quantitation (FRQ) mwachidule).Pakuyesa kwa NGS, tidapeza kuti FRQ ya zitsanzo za RNA imalumikizana kwambiri ndi kuchuluka kwa data.Pazitsanzo zonse zazikulu kuposa 0.2ng/uL FRQ, osachepera 70% ya zowerengera zimatha kuphimba bwino zotsatizana (Chithunzi 4).

Chithunzi 4, mtengo wa FRQ wodziwika ndi njira ya fluorescence yochuluka ili ndi mgwirizano wapamwamba kwambiri (R2> 0.9) ndi deta yothandiza yomwe imapezeka mu kuyesa kwa NGS.Mzere wofiira ndi mtengo wa FRQ wofanana ndi 0.2 ng/uL (log10 = -0.7).【4】

Kuphatikiza pakugwiritsa ntchito zitsanzo za FFPE, njira yeniyeni ya PCR yowerengera nthawi yeniyeni imathanso kuyang'anira zoletsa mu zitsanzo.Titha kuwonjezera zitsanzo kuti ziwonekere mumayendedwe ndi Internal Positive Control (IPC) ndi Assay yake, kenako ndikuchita fluorescence quantification kuti tipeze mtengo wa Ct.Ngati mtengo wa Ct ukutsalira kumbuyo kwa mtengo wa Ct muzotsatira zopanda chitsanzo, zimasonyeza kuti choletsa chilipo mu chitsanzocho ndipo chimalepheretsa kukulitsa mphamvu pakuchitapo kanthu.

05 Njira ya utoto wa Qubit fulorosenti

Qubit Fluorometer ndiye kachipangizo kakang'ono kamene kamagwiritsidwa ntchito kwambiri pozindikira kuti asidi a nucleic acid ndi kuyera, ndi yosavuta kugwiritsa ntchito ndipo amapezeka pafupifupi mu labotale iliyonse yazachilengedwe.Imawerengera molondola kuchuluka kwa nucleic acid pozindikira ndi nucleic acid-binding fluorescent dye (Qubit discovery reagent).Qubit ili ndi chidwi chachikulu komanso tsatanetsatane, ndipo imatha kuwerengera RNA mpaka pg/µL.Kuphatikiza pa luso lodziwika bwino lowerengera molondola kuchuluka kwa ma nucleic acid, mtundu waposachedwa wa Thermo Fisher, Qubit 4.0, ungathenso kuzindikira kukhulupirika kwa RNA.Dongosolo lozindikira la Qubit 4.0's RNA (RNA IQ Assay) limazindikira kukhulupirika kwa RNA pozindikira nthawi imodzi mitundu iwiri ya fulorosenti.Mitundu iwiriyi ya fulorosenti imatha kumangiriza tizidutswa tating'onoting'ono ta RNA, motsatana.Mitundu iwiri ya fulorosenti imasonyeza gawo la zidutswa zazikulu za RNA mu chitsanzo, ndipo kuchokera apa mtengo wa IQ (Ufulu ndi Ubwino) woimira khalidwe la RNA ukhoza kuwerengedwa.Mtengo wa IQ umagwira ntchito ku zitsanzo zonse za FFPE komanso zomwe si za FFPE, ndipo zimakhudza kwambiri mtundu wotsatizana.Kutengera zoyeserera za NGS monga mwachitsanzo, mu kuyesa kwa RNA-Seq komwe kunachitika pa nsanja ya Ion torrent™, zitsanzo zambiri zokhala ndi ma IQ apamwamba kuposa 4 zinali ndi zowerengera zogwira mtima 50% (Chithunzi 5).Poyerekeza ndi njira zodziwira zomwe tazitchula pamwambapa, Qubit IQ Assay sikuti ndi yabwino kwambiri kuti igwire ntchito ndipo imatenga nthawi yochepa (mkati mwa mphindi zisanu), komanso imakhala ndi mgwirizano waukulu pakati pa mtengo wa IQ woyezedwa ndi khalidwe la deta la zoyesera zapansi.

Chithunzi 5, pali kulumikizana kwakukulu pakati pa mtengo wa Qubit RNA IQ ndi kuwerengedwa kwa mapu a RNA-Seq.【5】

Kupyolera muzomwe zili pamwambazi, ndikukhulupirira kuti aliyense ali ndi chidziwitso chokwanira cha njira zosiyanasiyana zoyendetsera khalidwe la RNA.Pochita, mutha kusankhanjira yofananira molingana ndi mtundu wa zitsanzo ndi zida zomwe zilipo.Pokhapokha poyang'anira ubwino wa RNA bwino tingapewe kulephera kwa mayesero omwe amabwera chifukwa cha khalidwe lopanda chitsanzo, motero timasunga nthawi yamtengo wapatali, mphamvu ndi mtengo.

Zogulitsa:

Animal Total RNA Isolation Kit

Cell Total RNA Isolation Kit

maumboni

【1】Schroeder, A., Mueller, O., Stocker, S. et al.RIN: Nambala yodalirika ya RNA yogawira umphumphu ku miyeso ya RNA.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, Ma Metrics Owonjezera Apamwamba Owunika RNA Ochokera ku Archival Formalin-Fixed Paraffin-Embedded Tissue Samples, Toxicological Sciences, Volume 3720, August 3720, Issue 37, Ishttps://doi.org/10.1093/toxsci/