Kwaziwa kahle ukuthi enkolelweni emaphakathi, i-RNA ingumxhumanisi obhaliwe phakathi kwe-DNA nokuveza amaprotheni.Uma kuqhathaniswa nokutholwa kwe-DNA, ukutholwa kwe-RNA kungabonisa ngokunembile isisho sofuzo ezintweni eziphilayo.Ukuhlola okubandakanya i-RNA kuhlanganisa: i-qRT-PCR, i-RNA-Seq, nokutholwa kofuzo oluhlanganisiwe, njll. Ngokusekelwe kuzici ze-RNA ngokwayo (indandatho kashukela ye-RNA ineqembu elilodwa lamahhala le-hydroxyl kuneringi likashukela le-DNA), kuhlanganiswe nenani elikhulu lama-RNases endaweni, i-RNA ayizinzile futhi kulula ukucekelwa phansi kune-DNA.Udoti ungaphakathi, udoti uphumile, uma ikhwalithi ye-RNA ingeyinhle, khona-ke imiphumela yokuhlola kufanele ingagculisi, iboniswe ngokuqondile njengedatha engalungile noma ukuphindaphinda okubi.Ngakho-ke, kufanele kuqashelwe kakhulu ekucutshungulweni kwe-RNA, futhi isixhumanisi sokulawulwa kwekhwalithi sibaluleke kakhulu ukuze kuqinisekiswe ukunemba nokunemba kwedatha yokuhlola elandelayo.

Ukulawulwa kwekhwalithi ye-RNA, ngokuvamile kunezindlela ezilandelayo ezivame ukusetshenziswa:

• I-Spectrophotometry
• i-agarose gel electrophoresis
• I-Agilent Bioanalyzer
• real-time fluorescent quantitative PCR
• Indlela yokudaya ye-Qubit Fluorescent

01 I-Spectrophotometry

I-RNA ihlanganise amabhondi aphindwe kabili futhi inenani eliphakeme lokumuncwa kubude begagasi obungu-260nm.Ngokomthetho we-Lambert-Beer, singabala ukugxiliswa kwe-RNA kusukela ekumunceni okuphezulu ku-260nm.Ngaphezu kwalokho, singakwazi futhi ukubala ubumsulwa be-RNA ngokwesilinganiso sama-260nm, 280nm kanye neziqongo zokumunca ezingama-230nm.I-280nm kanye ne-230nm yiziqongo zokumunca amaprotheni nama-molecule amancane, ngokulandelana.Isilinganiso se-A260/A280 kanye ne-A260/A230 yokuhlanzeka kwe-RNA okufanelekayo kufanele sibe sikhulu kuno-2. Uma singaphansi kuka-2, kusho ukuthi kukhona amaprotheni noma i-molecule encane kusampula ye-RNA futhi idinga ukuhlanzwa futhi.Imithombo yokungcola izothinta ukuhlola okwehlayo, okufana nokuvimbela ukusebenza kahle kokukhulisa ukusabela kwe-PCR, okuholela emiphumeleni yobuningi enganembile.Ukuhlanzeka kwe-RNA kunethonya elikhulu emiphumeleni elandelayo, ngakho-ke i-spectrophotometry ngokuvamile iyisixhumanisi sokulawula ikhwalithi esibalulekile esinyathelweni sokuqala sokuhlolwa kwe-nucleic acid.

Umfanekiso 1. I-RNA/DNA Absorption Spectrum Ejwayelekile

02 Ijeli ye-Agarose electrophoresis

Ngaphezu kobumsulwa, ubuqotho be-RNA futhi bungenye yezinkomba ezibalulekile zokwahlulela ikhwalithi ye-RNA.Ukucekelwa phansi kwe-RNA kuzoholela enanini elikhulu lezingcezu ezimfushane kusampula, ngakho inani lezingcezu ze-RNA ezingatholwa ngempumelelo futhi zembozwe ukulandelana kwereferensi kuzoncishiswa.Ubuqotho be-RNA bungahlolwa nge-electrophoresis yenani le-RNA kujeli ye-agarose engu-1%.Le ndlela ingalungiselela ijeli ngokwakho, noma isebenzise i-E-Gel™ System eseyakhiwe ngaphambili ukuze ihlole ubuqotho.Ngaphezu kuka-80% wesamba se-RNA i-ribosomal RNA, iningi layo elakhiwe ama-28S kanye ne-18S rRNA (ezinhlelweni ezincelisayo).Ikhwalithi enhle ye-RNA izobonisa amabha amabili agqamile asobala, okungamabha agqamile angu-28S no-18S, ngokulandelana, ku-5 Kb no-2 Kb, futhi isilinganiso sizoba seduze no-2:1.Uma isesimweni sokusabalalisa, kusho ukuthi isampula ye-RNA kungenzeka yehlisiwe, futhi kuyanconywa ukuthi kusetshenziswe indlela echazwe kamuva ukuze kuqhutshekwe nokuhlola ikhwalithi ye-RNA.

Umfanekiso 2. Ukuqhathaniswa kwe-degraded (umzila 2) kanye ne-RNA eqinile (umzila 3) ku-agarose gel electrophoresis

03 I-Agilent Bioanalyzer

Ngaphezu kwendlela ye-agarose gel electrophoresis echazwe ngenhla, engasisiza ukuthi sibone ubuqotho be-RNA kalula futhi ngokushesha, singasebenzisa i-Agilent bioanalyzer ukuze sinqume ubuqotho be-RNA.Isebenzisa inhlanganisela ye-microfluidics, i-capillary electrophoresis, ne-fluorescence ukuhlola ukugxila kwe-RNA nobuqotho.Ngokusebenzisa i-algorithm eyakhelwe ngaphakathi ukuze ihlaziye iphrofayela yesampula ye-RNA, i-Agilent bioanalyzer ingabala inani lobuqotho le-RNA eliyinkomba, Inombolo Yobuqotho ye-RNA (ngemuva kwalokhu ebizwa ngokuthi i-RIN) [1].Uma likhulu inani le-RIN, liphakamisa ubuqotho be-RNA (u-1 wehliswe ngokwedlulele, u-10 uphelele kakhulu).Okunye ukuhlola okubandakanya i-RNA kuphakamisa ukusebenzisa i-RIN njengepharamitha yokuhlola ikhwalithi.Ukuthatha ukuhlolwa kokulandelana komkhiqizo ophezulu (okubizwa kamuva ngokuthi yi-NGS) njengesibonelo, imihlahlandlela ye-Oncomine™ Human Immune Repertoire, esetshenziselwa ukuthola ama-B cell kanye nama-T cell antigen receptors ochungechungeni lwephaneli ye-Thermo Fisher's Oncomine, iphakamisa ukuthi amasampuli anamanani e-RIN angaphezu kuka-4, Ukufundwa okuphumelelayo Kakhudlwana nama-clones kungenziwa (Figure 3).Kunobubanzi obuhlukile obunconyiwe bamaphaneli ahlukene, futhi ngokuvamile i-RIN ephakeme ingaletha idatha esebenza kahle kakhulu.

Umfanekiso 3, ekuhlolweni kwe-Oncomine™ Human Immune Repertoire, amasampula ane-RIN angaphezu kuka-4 angathola ukufundwa okusebenza kahle kakhulu nama-T cell clones.【2】

Nokho, inani le-RIN nalo linemikhawulo ethile.Nakuba i-RIN inokuhlobana okuphezulu nekhwalithi yedatha yokuhlola ye-NGS, ayiwafanele amasampuli e-FFPE.Amasampula e-FFPE alashwe ngamakhemikhali isikhathi eside, futhi i-RNA ekhishiwe ngokuvamile inenani eliphansi le-RIN.Nokho, lokhu akusho ukuthi idatha esebenzayo yokuhlolwa kufanele ingagculisi.Ukuhlola ngokunembile ikhwalithi yamasampuli e-FFPE, sidinga ukusebenzisa izilinganiso ngaphandle kwe-RIN.Ngokungeziwe ku-RIN, i-Agilent bioanalyzer ingaphinda ibale inani le-DV200 njengepharamitha yokuhlola yekhwalithi ye-RNA.I-DV200 ipharamitha ebala ingxenye yezingcezu ezinkulu kuno-200 bp kusampula ye-RNA.I-DV200 iyinkomba engcono yekhwalithi yesampula ye-FFPE kune-RIN.Ku-RNA ekhishwe yi-FFPE, inokuhlobana okuphezulu kakhulu nenani lezakhi zofuzo ezingatholwa ngempumelelo kanye nokwehlukahlukana kwezakhi zofuzo [3].Nakuba i-DV200 ingakwazi ukuvala isikhala ekutholweni kwekhwalithi ye-FFPE, i-Agilent bioanalyzer namanje ayikwazi ukuhlaziya izinkinga zekhwalithi kumasampuli e-RNA, okuhlanganisa ukuthi akhona yini ama-inhibitor kumasampuli.Ama-inhibitors ngokwawo angathinta ukusebenza kahle kokukhulisa izivivinyo ezingezansi futhi anciphise inani ledatha ewusizo.Ukuze sazi ukuthi ingabe ikhona yini inhibitor kusampula, singasebenzisa indlela ye-PCR yesikhathi sangempela ye-fluorescent echazwe ngokulandelayo.

04 real-time fluorescent quantitative PCR

Indlela ye-PCR yobuningi be-fluorescent yesikhathi sangempela ayikwazi nje ukubona ama-inhibitor kusampula, kodwa futhi ibonise ngokunembile ikhwalithi ye-RNA kusampula ye-FFPE.Uma kuqhathaniswa nabahlaziyi bebhayoloji be-Agilent, amathuluzi omthamo we-fluorescence wesikhathi sangempela adume kakhulu kumalabhorethri amakhulu ebhayoloji ngenxa yokusetshenziswa kwawo okubanzi.Ukuze sihlole ikhwalithi yamasampuli e-RNA, sidinga kuphela ukuthenga noma ukulungisa ama-primer probe ofuzo lwangaphakathi oluyisethenjwa, njenge-GUSB (Cat no. Hs00939627).Ngokusebenzisa le sethi yeziqalo, ama-probes kanye namazinga (ingqikithi ye-RNA yokugxilisa ingqondo okwaziwayo) ukuze kwenziwe izivivinyo zenani eliphelele, ukugxiliswa kwesiqephu se-RNA esisebenzayo singabalwa njengezinga lokuhlola lekhwalithi ye-RNA (Functional RNA Quantitation (FRQ) ngamafuphi).Ekuhlolweni kwe-NGS, sithole ukuthi i-FRQ yamasampuli e-RNA inokuhlobana okuphezulu kakhulu nevolumu yedatha esebenzayo.Kuwo wonke amasampuli angaphezu kuka-0.2ng/uL FRQ, okungenani u-70% ofundiwe angavala ngempumelelo ukulandelana kwereferensi (Umfanekiso 4).

Umfanekiso wesi-4, inani le-FRQ elitholwe indlela yobuningi be-fluorescence linokuxhumana okuphezulu kakhulu (R2>0.9) nedatha esebenzayo etholwe ekuhlolweni kwe-NGS.Umugqa obomvu uyinani le-FRQ elilingana no-0.2 ng/uL (log10 = -0.7).【4】

Ngaphezu kokusebenza kumasampuli e-FFPE, indlela ye-PCR yobuningi besikhathi sangempela ingaphinda igade ama-inhibitor kumasampuli.Singangeza isampula ukuze itholwe kusistimu yokusabela nge-Internal Positive Control (IPC) kanye ne-Assay yayo, bese senze ukulinganisa kwe-fluorescence ukuze sithole inani le-Ct.Uma inani le-Ct lisala ngemuva kwevelu ye-Ct ekuphenduleni okungenasampula, kubonisa ukuthi inhibitor ikhona kusampula futhi ivimbela ukusebenza kahle kokukhulisa ekuphenduleni.

05 Indlela yokudaya ye-Qubit fluorescent

I-Qubit Fluorometer iyisisetshenziswa esincane esivame ukusetshenziswa kakhulu sokugxilisa i-nucleic acid nokuhlonza ubumsulwa, okulula ukusebenza futhi esikhona cishe kuyo yonke ilabhorethri yebhayoloji yamangqamuzana.Ibala ngokunembile ukugcwala kwe-nucleic acid ngokuthola kanye nodayi we-fluorescent obopha i-nucleic acid (i-Qubit discover reagent).I-Qubit inokuzwela okuphezulu nokucaciswa, futhi ingakwazi ukulinganisa ngokunembile i-RNA yehle iye ku-pg/µL ukugxila.Ngaphezu kwekhono elaziwayo lokulinganisa ngokunembile ukugcwala kwe-nucleic acid, imodeli entsha yakamuva ye-Thermo Fisher, i-Qubit 4.0, ingakwazi futhi ukubona ubuqotho be-RNA.Isistimu yokuthola i-Qubit 4.0′s RNA (RNA IQ Assay) ithola ubuqotho be-RNA ngokuthola ngesikhathi esisodwa odayi ababili abakhethekile befluorescent.Laba odayi ababili be-fluorescent bangabopha izingcezu ezinkulu nezingcezu ezincane ze-RNA, ngokulandelana.Laba odayi ababili befluorescent babonisa ingxenye yezingcezu ezinkulu ze-RNA kusampula, futhi kusukela kulokhu inani le-IQ (Ubuqotho Nekhwalithi) elimelela ikhwalithi ye-RNA lingabalwa.Inani le-IQ lisebenza kuwo womabili amasampuli e-FFPE nangewona awe-FFPE, futhi linomthelela omkhulu kukhwalithi yokulandelana elandelayo.Sithatha izivivinyo ze-NGS njengesibonelo, ekuhlolweni kokuhlolwa kwe-RNA-Seq okwenziwe kuplathifomu ye-Ion torrent™, amasampuli amaningi anamanani e-IQ angaphezu kuka-4 abe nokufundwa okusebenzayo okungenani okungu-50% (Umfanekiso 5).Uma kuqhathaniswa nezindlela zokuthola ezibalulwe ngenhla, i-Qubit IQ Assay ayilula nje kuphela ukusebenza futhi ithatha isikhathi esincane (phakathi kwemizuzu emihlanu), kodwa futhi inokuhlobana okuhle phakathi kwevelu yepharamitha elinganisiwe ye-IQ kanye nekhwalithi yedatha yokuhlolwa komfula.

Umfanekiso 5, kukhona ukuhlobana okukhulu phakathi kwevelu ye-Qubit RNA IQ kanye nokufundwa kwemephu kwe-RNA-Seq.【5】

Ngalesi singeniso esingenhla, ngikholwa ukuthi wonke umuntu unokuqonda okwanele kwezindlela ezahlukene zokulawula ikhwalithi ye-RNA.Ngokwenza, ungakhethaindlela ehambisanayo ngokohlobo lwesampula namathuluzi akhona.Kuphela ngokulawula ikhwalithi ye-RNA kahle singagwema ukwehluleka kokuhlolwa okulandelayo okubangelwa ikhwalithi yesampula ephansi, ngaleyo ndlela songe isikhathi esiyigugu, amandla kanye nezindleko.

Imikhiqizo eyinkomba:

Ikhithi Yokuhlukanisa Yezilwane Ephelele ye-RNA

Ikhithi Yokuzihlukanisa Yeseli Ye-RNA

izinkomba

【1】Schroeder, A., Mueller, O., Stocker, S. et al.I-RIN: inombolo yobuqotho ye-RNA yokunikeza amanani obuqotho kuzilinganiso ze-RNA.I-BMC Molecular Biol 7, 3 (2006).https:// doi .org/10.1186/1471-21 99-7-3

【2】Umhlahlandlela Womsebenzisi we-Oncomine Human Immune Repertoire (Pub. No. MAN0017438 Rev. C.0).

【3】ULeah C Wehmas, uCharles E Wood, uBrian N Chorley, uCarole L Yauk, uGail M Nelson, uSusan D Hester, Amamethrikhi Ekhwalithi Ethuthukisiwe Wokuhlola i-RNA Esuselwa Ku-Archival Formalin-Fixed Paraffin-Embedded Tissue Samples, Toxicological Sciences, Volume 3720, Agasti 320, Issue 3, Ishttps://doi.org/10.1093/toxsci/