Hoa tsebahala hore thutong e bohareng, RNA ke mokena-lipakeng oa transcriptional lipakeng tsa DNA le protheine expression.Ha ho bapisoa le ho sibolloa ha DNA, ho sibolloa ha RNA ho ka bonahatsa ka mokhoa o nepahetseng polelo ea liphatsa tsa lefutso linthong tse phelang.Liteko tse amanang le RNA li kenyelletsa: qRT-PCR, RNA-Seq, le ho lemoha liphatsa tsa lefutso tsa fusion, joalo-joalo Ho itšetlehile ka litšobotsi tsa RNA ka boeona (ring ea tsoekere ea RNA e na le sehlopha se le seng sa mahala sa hydroxyl ho feta sa tsoekere ea DNA), hammoho le palo e kholo ea RNases tikolohong, RNA e tsitsitse ebile e bonolo ho senyeha ho feta DNA.Litšila ka har'a, litšila li tsoa, ​​haeba boleng ba RNA bo se botle, joale liphetho tsa liteko li tlameha ho ba tse sa khotsofatseng, tse bonts'itsoeng ka ho khetheha e le data e sa nepahalang kapa ho pheta-pheta hampe.Ka hona, tlhokomelo e eketsehileng e lokela ho lefshoa ts'ebetsong ea RNA, 'me sehokelo sa taolo ea boleng le sona se bohlokoa haholo ho netefatsa ho nepahala le ho nepahala ha data ea liteko tse latelang.

Bakeng sa taolo ea boleng ba RNA, hangata ho na le mekhoa e latelang e sebelisoang hangata:

• Spectrophotometry
• agarose gel electrophoresis
• Agilent Bioanalyzer
• PCR ea nako ea nnete ea fluorescent
• Mokhoa oa ho daea oa fluorescent

01 Spectrophotometry

RNA e hokahane le li-bond tse peli mme e na le tlhoro ea ho monya ka bolelele ba 260nm.Ho ea ka molao oa Lambert-Beer, re ka bala mahloriso a RNA ho tloha tlhorong ea ho monya ho 260nm.Ntle le moo, re ka boela ra bala bohloeki ba RNA ho latela karo-karolelano ea 260nm, 280nm le 230nm litlhoro tsa ho monya.280nm le 230nm ke litlhoro tsa ho monya ha liprotheine le limolek'hule tse nyane, ka ho latellana.Karo-karolelano ea A260 / A280 le A260 / A230 ea bohloeki ba RNA e tšoanelehang e lokela ho ba kholo ho feta 2. Haeba e ka tlaase ho 2, ho bolela hore ho na le tšilafalo ea protheine kapa molek'hule e nyenyane sampuling ea RNA 'me e hloka ho hloekisoa hape.Mehloli ea tšilafalo e tla ama liteko tse tlase, joalo ka ho thibela ho sebetsa hantle ha karabelo ea PCR, ho baka liphetho tse sa nepahalang tsa palo.Bohloeki ba RNA bo na le tšusumetso e kholo liphethong tse latelang, kahoo spectrophotometry ka kakaretso ke sehokelo sa bohlokoa sa taolo ea boleng mohatong oa pele oa liteko tsa nucleic acid.

Setšoantšo sa 1. Tloaelo ea RNA / DNA Absorption Spectrum

02 Agarose gel electrophoresis

Ntle le bohloeki, botšepehi ba RNA hape ke e 'ngoe ea matšoao a bohlokoa bakeng sa ho ahlola boleng ba RNA.Ho senyeha ha RNA ho tla lebisa palo e kholo ea likaroloana tse khutšoanyane sampuling, kahoo palo ea likhechana tsa RNA tse ka fumanoang ka katleho le ho koaheloa ke tatellano ea litšupiso li tla fokotseha.Botšepehi ba RNA bo ka hlahlojoa ka electrophoresis ea kakaretso ea RNA ho gel ea agarose ea 1%.Mokhoa ona o ka iketsetsa gel ka bouena, kapa oa sebelisa E-Gel™ System e entsoeng esale pele bakeng sa tlhahlobo ea botšepehi.Ho feta 80% ea kakaretso ea RNA ke ribosomal RNA, eo bongata ba eona e entsoeng ka 28S le 18S rRNA (litsamaisong tsa mammalian).RNA ea boleng bo botle e tla bontša mekoallo e 'meli e hlakileng e khanyang, e leng 28S le 18S mekoallo e khanyang, ka ho latellana, ho 5 Kb le 2 Kb,' me karo-karolelano e tla ba haufi le 2: 1.Haeba e le boemong bo fapaneng, ho bolela hore sampuli ea RNA e ka 'na ea senyeha,' me ho khothalletsoa ho sebelisa mokhoa o hlalositsoeng hamorao ho hlahloba boleng ba RNA ho ea pele.

Setšoantšo sa 2. Papiso ea ho senyeha (lane ea 2) le RNA e tsitsitseng (lane ea 3) ho agarose gel electrophoresis

03 Agilent Bioanalyzer

Ntle le mokhoa oa agarose gel electrophoresis o hlalositsoeng ka holimo, o ka re thusang ho tseba botšepehi ba RNA habonolo le ka potlako, re ka boela ra sebelisa Agilent bioanalyzer ho fumana botšepehi ba RNA.E sebelisa motsoako oa microfluidics, capillary electrophoresis, le fluorescence ho hlahloba mahloriso a RNA le botšepehi.Ka ho sebelisa algorithm e hahelletsoeng ho sekaseka profil ea sampole ea RNA, Agilent bioanalyzer e ka bala boleng ba bots'epehi ba RNA, Nomoro ea Botšepehi ea RNA (eo ka mor'a moo e bitsoang RIN) [1].Ha boleng ba RIN bo le boholo, botšepehi ba RNA bo phahame haholo (1 e senyehile haholo, 10 ke eona e feletseng ka ho fetisisa).Liteko tse ling tse amang RNA li fana ka maikutlo a ho sebelisa RIN joalo ka paramethara bakeng sa tlhahlobo ea boleng.Ho nka liteko tse phahameng tsa tatellano (tseo ka morao ho moo ho thoeng ke NGS) e le mohlala, litataiso tsa Oncomine™ Human Immune Repertoire, e sebelisetsoang ho lemoha B cell le T cell antigen receptors ho Thermo Fisher's Oncomine panel series, e fana ka maikutlo a hore disampole tse nang le boleng ba RIN tse kholo ho feta 4, Litekanyo tse sebetsang haholoanyane le li-clones li ka ba 3.Ho na le mekhahlelo e fapaneng e khothaletsoang bakeng sa liphanele tse fapaneng, 'me hangata RIN e phahameng e ka tlisa data e sebetsang haholoanyane.

Setšoantšo sa 3, litekong tsa Oncomine™ Human Immune Repertoire, lisampole tse nang le RIN e kholo ho feta 4 li ka bona ho baloa ho hoholo le li-clones tsa T cell.【2】

Leha ho le joalo, boleng ba RIN bo boetse bo na le mefokolo e itseng.Leha RIN e na le kamano e phahameng le boleng ba data ea liteko ea NGS, ha e ea lokela disampole tsa FFPE.Mehlala ea FFPE e 'nile ea phekoloa ka lik'hemik'hale nako e telele,' me RNA e nkiloeng ka kakaretso e na le boleng bo tlaase ba RIN.Leha ho le joalo, sena ha se bolele hore data e sebetsang ea teko e tlameha ho ba e sa khotsofatseng.Ho hlahloba ka nepo boleng ba disampole tsa FFPE, re hloka ho sebelisa litekanyo tse ling ntle le RIN.Ntle le RIN, Agilent bioanalyzer e ka boela ea bala boleng ba DV200 e le parameter ea tlhahlobo ea boleng ba RNA.DV200 ke paramethara e lekanyang karolo ea likhechana tse kholo ho feta 200 bp sampole ea RNA.DV200 ke sesupo se betere sa boleng ba sampole ea FFPE ho feta RIN.Bakeng sa RNA e ntšitsoeng ke FFPE, e na le kamano e phahameng haholo le palo ea liphatsa tsa lefutso tse ka fumanoang ka katleho le mefuta-futa ea liphatsa tsa lefutso [3].Le hoja DV200 e ka etsa bofokoli ba ho lemoha boleng ba FFPE, Agilent bioanalyzer e ntse e sitoa ho sekaseka mathata a boleng lisampoleng tsa RNA, ho kenyelletsa le hore na ho na le li-inhibitors lisampole.Li-inhibitors ka botsona li ka ama katleho ea ho holisa liteko tse tlase le ho fokotsa bongata ba data e sebetsang.Ho tseba hore na ho na le inhibitor sampoleng, re ka sebelisa mokhoa oa 'nete oa nako ea fluorescent quantitative PCR o hlalositsoeng ka mor'a moo.

04 PCR ea nako ea nnete ea fluorescent

Mokhoa oa 'nete oa nako ea fluorescent quantitative PCR ha o khone ho lemoha li-inhibitor sampole feela, empa hape o bonts'a boleng ba RNA sampole ea FFPE ka nepo.Ha ho bapisoa le Agilent biological analyzers, lisebelisoa tsa nako ea nnete tsa fluorescence quantitative li tsebahala haholo lilaboratoring tse kholo tsa baeloji ka lebaka la ts'ebeliso ea tsona e pharalletseng.Ho leka boleng ba lisampole tsa RNA, re hloka feela ho reka kapa ho lokisa li-primer probes bakeng sa liphatsa tsa lefutso tsa ka hare, tse kang GUSB (Cat no. Hs00939627).Ka ho sebelisa sete sena sa li-primers, probes le standards (kakaretso ea RNA ea mahloriso a tsejoang) ho etsa liteko tse felletseng tsa bongata, mohopolo o sebetsang oa sekhechana sa RNA o ka baloa e le tekanyetso ea tlhahlobo ea boleng ba RNA (Functional RNA Quantitation (FRQ) ka nako e khuts'oane).Tekong ea NGS, re fumane hore FRQ ea lisampole tsa RNA e na le khokahano e phahameng haholo le bophahamo ba data bo sebetsang.Bakeng sa disampole tsohle tse kholo ho feta 0.2ng/uL FRQ, bonyane 70% ea tse baloang e ka koahela ka nepo tatellano ea litšupiso (Setšoantšo sa 4).

Setšoantšo sa 4, boleng ba FRQ bo fumanoeng ka mokhoa oa fluorescence quantitative o na le kamano e phahameng haholo (R2> 0.9) le boitsebiso bo sebetsang bo fumanoeng tekong ea NGS.Mohala o mofubelu ke boleng ba FRQ bo lekanang le 0.2 ng/uL (log10 = -0.7).【4】

Ntle le ho sebetsa ho disampole tsa FFPE, mokhoa oa 'nete oa PCR oa nako ea nnete o ka boela oa beha leihlo li-inhibitor ka lisampole.Re ka eketsa sampole e tla bonoa ho sistimi ea karabelo ka Internal Positive Control (IPC) le Assay ea eona, ebe re etsa fluorescence quantification ho fumana boleng ba Ct.Haeba boleng ba Ct bo salla ka morao ho boleng ba Ct karabelong e se nang mohlala, e bontša hore inhibitor e teng sampuling mme e thibela katleho ea amplification karabelong.

05 Mokhoa oa ho daea oa fluorescent oa Qubit

Qubit Fluorometer ke sesebelisoa se senyenyane se sebelisoang ka ho fetesisa bakeng sa ho tsepamisa mohopolo oa nucleic acid le ho lemoha bohloeki, seo ho leng bonolo ho se sebetsa ebile se teng hoo e ka bang laboratoring e 'ngoe le e 'ngoe ea baeloji ea limolek'hule.E lekanya ka nepo bongata ba asiti ea nucleic ka ho bona le dae ea fluorescent e tlamang nucleic acid (Qubit discovery reagent).Qubit e na le kutlo e phahameng le ho hlaka, 'me e khona ho lekanya RNA ka nepo ho fihla ho pg/µL ea mahloriso.Ntle le bokhoni bo tsebahalang ba ho lekanya ka nepo khatello ea nucleic acid, mofuta o mocha oa morao-rao oa Thermo Fisher, Qubit 4.0, o ka boela oa lemoha botšepehi ba RNA.Sistimi ea ho lemoha ea Qubit 4.0's RNA (RNA IQ Assay) e lemoha botšepehi ba RNA ka ho lemoha ka nako e le 'ngoe lidae tse peli tse khethehileng tsa fluorescent.Lidae tsena tse peli tsa fluorescent li ka kopanya likaroloana tse kholo le likaroloana tse nyane tsa RNA, ka ho latellana.Lidae tsena tse peli tsa fluorescent li bonts'a karolo ea likaroloana tse kholo tsa RNA sampole, 'me ho tloha ho sena boleng ba IQ (Botšepehi le Boleng) bo emelang boleng ba RNA bo ka baloa.Boleng ba IQ bo sebetsa ho disampole tsa FFPE le tseo eseng tsa FFPE, 'me li na le tšusumetso e kholo ho boleng ba tatellano e latelang.Ho nka liteko tsa NGS e le mohlala, litekong tsa liteko tsa RNA-Seq tse entsoeng sethaleng sa Ion torrent™, lisampole tse ngata tse nang le litekanyetso tsa IQ tse kholo ho feta 4 li bile le bonyane 50% ea ho bala hantle (Setšoantšo sa 5).Ha ho bapisoa le mekhoa ea ho lemoha e boletsoeng ka holimo, Qubit IQ Assay ha e bonolo feela ho sebetsa mme e nka nako e fokolang (ka metsotso e mehlano), empa e boetse e na le kamano e kholo pakeng tsa boleng ba parameter ea IQ e lekantsoeng le boleng ba data ba liteko tse tlaase.

Setšoantšo sa 5, ho na le kamano e kholo lipakeng tsa boleng ba Qubit RNA IQ le 'mapa oa RNA-Seq.【5】

Ka kenyelletso e ka holimo, ke lumela hore e mong le e mong o na le kutloisiso e lekaneng ea mekhoa e fapaneng ea ho laola boleng ba RNA.Ka ts'ebetso, u ka khethamokhoa o tsamaellanang ho latela mofuta oa mohlala le lisebelisoa tse teng.Ke feela ka ho laola boleng ba RNA hantle re ka qobang ho hlōleha ha liteko tse latelang tse bakoang ke boleng bo bobe ba sampole, kahoo re boloka nako ea bohlokoa, matla le litšenyehelo.

Lihlahisoa tsa litšupiso:

Animal Total RNA Isolation Kit

Cell Total RNA Isolation Kit

litšupiso

【1】Schroeder, A., Mueller, O., Stocker, S. et al.RIN: nomoro ea botšepehi ea RNA bakeng sa ho abela boleng ba botšepehi ho litekanyo tsa RNA.BMC Molek'hule 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, Metrics e Ntlafalitsoeng ea Boleng bakeng sa Tlhahlobo ea RNA e Nkiloeng ho Archival Formalin-Fixed Paraffin-Embedded Tissue Samples, Toxicological Sciences, Volume 3, 1729, August 3, 7https://doi.org/10.1093/toxsci/