Ua ʻike ʻia ma ka dogma waena, ʻo RNA ka transcriptional mediator ma waena o ka DNA a me ka hōʻike protein.Ke hoʻohālikelike ʻia me ka ʻike ʻana i ka DNA, hiki i ka ʻike ʻana o RNA ke hōʻike pono i ka hōʻike ʻana i nā mea ola.ʻO nā hoʻokolohua e pili ana i ka RNA: qRT-PCR, RNA-Seq, a me ka fusion gene detection, etc. Ma muli o nā hiʻohiʻona o RNA ponoʻī (ʻo ke apo kō o RNA he hoʻokahi hui hydroxyl manuahi ʻoi aʻe ma mua o ke apo kō o DNA), i hui pū ʻia me ka nui o nā RNases i ke kaiapuni, ʻoi aku ka paʻa ʻole o ka RNA a maʻalahi hoʻi e hoʻohaʻahaʻa ʻia ma mua o DNA.ʻO ka ʻōpala i loko, ʻōpala i waho, inā ʻaʻole maikaʻi ka maikaʻi o ka RNA, a laila pono ʻole nā ​​​​hopena hoʻokolohua, i hōʻike ʻia ma ke ʻano he ʻikepili pololei ʻole a i ʻole ka hana hou ʻana.No laila, ʻoi aku ka nui o ka nānā ʻana i ka hana ʻana o RNA, a ʻoi aku ka nui o ka loulou o ka mana maikaʻi e hōʻoia i ka pololei a me ka pololei o nā ʻikepili hoʻokolohua ma hope.

No ka hoʻomalu maikaʻi ʻana o RNA, aia nā ʻano hana maʻamau i hoʻohana ʻia:

• Spectrophotometry
• electrophoresis gel agarose
• Agilent Bioanalyzer
• ʻO ka PCR nui o ka fluorescent manawa maoli
• ʻO ke ʻano hoʻonanea ʻo Qubit fluorescent

01 ʻIkepili

Ua hoʻohui ʻo RNA i nā mea paʻa pālua a loaʻa iā ia kahi kiʻekiʻe absorption ma kahi hawewe o 260nm.Wahi a ke kānāwai o Lambert-Beer, hiki iā mākou ke helu i ka manaʻo RNA mai ka piko absorption ma 260nm.Eia kekahi, hiki iā mākou ke helu i ka maʻemaʻe o RNA e like me ka ratio o 260nm, 280nm a me 230nm absorption peaks.ʻO 280nm a me 230nm nā kiʻekiʻe absorption o nā protein a me nā molekole liʻiliʻi, kēlā me kēia.ʻO ka lakio o A260 / A280 a me A260 / A230 o ka maʻemaʻe RNA kūpono e ʻoi aku ka nui ma mua o 2. Inā ʻoi aku ka liʻiliʻi ma mua o 2, ʻo ia hoʻi, aia ka protein a i ʻole nā ​​mole liʻiliʻi contamination i ka hāpana RNA a pono e hoʻomaʻemaʻe hou.E hoʻopilikia nā kumu hoʻohaumia i nā hoʻokolohua o lalo, e like me ke kāohi ʻana i ka hoʻonui ʻana i ka pono o nā hopena PCR, e hopena i nā hopena quantitative pololei ʻole.He hopena nui ka maʻemaʻe o RNA i nā hopena ma hope, no laila, ʻo ka spectrophotometry ka mea maʻamau he loulou mana maikaʻi loa i ka hana mua i nā hoʻokolohua nucleic acid.

Kiʻi 1. RNA/DNA Absorption Spectrum maʻamau

02 Agarose gel electrophoresis

Ma waho aʻe o ka maʻemaʻe, ʻo ka pono o ka RNA kekahi o nā hōʻailona koʻikoʻi no ka hoʻoholo ʻana i ka maikaʻi o ka RNA.ʻO ka hoʻohaʻahaʻa ʻana o ka RNA e alakaʻi i ka nui o nā ʻāpana pōkole i ka hāpana, no laila e hoʻemi ʻia ka helu o nā ʻāpana RNA i hiki ke ʻike pono ʻia a uhi ʻia e ke kaʻina kuhikuhi.Hiki ke nānā ʻia ka pono RNA e ka electrophoresis o ka huina RNA ma kahi 1% agarose gel.Hiki i kēia ʻano hana ke hoʻonohonoho i ka gel iā ʻoe iho, a i ʻole e hoʻohana i ka Pūnaehana E-Gel™ prefabricated no ka hoʻāʻo pono ʻana.ʻOi aku ma mua o 80% o ka nui o ka RNA he ribosomal RNA, ʻo ka hapa nui o ia mau mea he 28S a me 18S rRNA (ma nā ʻōnaehana mammalian).E hōʻike ana ka RNA maikaʻi maikaʻi i ʻelua mau kaola ʻālohilohi, ʻo ia ka 28S a me 18S mau pahu kukui, ma 5 Kb a me 2 Kb, a ʻo ka ratio e kokoke kokoke i 2:1.Inā i loko o kahi moku'āina ākea, 'o ia ho'i, ua ho'oha'aha'a 'ia ka la'ana RNA, a ua paipai 'ia e ho'ohana i ke ala i wehewehe 'ia ma hope e ho'ā'o hou ai i ka maika'i o ka RNA.

Kiʻi 2. Ka hoʻohālikelike o ka degraded (lane 2) a me ka RNA paʻa (lane 3) ma ka electrophoresis gel agarose

03 Agilent Bioanalyzer

Ma waho aʻe o ke kaʻina electrophoresis gel agarose i hōʻike ʻia ma luna, hiki ke kōkua iā mākou e ʻike i ka pono o ka RNA maʻalahi a me ka wikiwiki, hiki iā mākou ke hoʻohana i ka bioanalyzer Agilent e hoʻoholo ai i ka pono o RNA.Hoʻohana ia i ka hui pū ʻana o microfluidics, capillary electrophoresis, a me fluorescence e loiloi i ka ʻike a me ka pololei o RNA.Ma ka hoʻohana ʻana i ka algorithm i kūkulu ʻia no ka nānā ʻana i ka ʻaoʻao o ka laʻana RNA, hiki i ka Agilent bioanalyzer ke helu i kahi waiwai pono RNA kuhikuhi, RNA Integrity Number (i kapa ʻia ʻo RIN) [1].ʻO ka nui o ka waiwai o RIN, ʻoi aku ka kiʻekiʻe o ka pono o ka RNA (1 ua hoʻohaʻahaʻa loa ʻia, ʻo 10 ka mea piha loa).Manaʻo kekahi mau hoʻokolohua e pili ana i ka RNA e hoʻohana i ka RIN ma ke ʻano he ʻāpana no ka loiloi maikaʻi.ʻO ka lawe ʻana i nā hoʻokolohua hoʻokolohua kiʻekiʻe-throughput (ma hope aku i kapa ʻia ʻo NGS) ma ke ʻano he laʻana, nā alakaʻi o Oncomine ™ Human Immune Repertoire, i hoʻohana ʻia e ʻike ai i nā cell B a me T cell antigen receptors ma Thermo Fisher's Oncomine panel series, manaʻo ʻia nā laʻana me nā waiwai RIN i ʻoi aku ma mua o 4, Hiki ke ana ʻia nā heluhelu a me nā clones ʻoi aku ka maikaʻi (Figure 3).Aia nā pae ʻokoʻa i manaʻo ʻia no nā panela like ʻole, a pinepine ka RIN kiʻekiʻe e hiki ke lawe mai i ka ʻikepili ʻoi aku ka maikaʻi.

Kiʻi 3, ma nā hoʻokolohua Oncomine™ Human Immune Repertoire, hiki i nā laʻana me RIN ʻoi aku ma mua o 4 ke ʻike i nā heluhelu ʻoi aku ka maikaʻi a me nā clones T cell.【2】

Eia naʻe, loaʻa i ka waiwai RIN kekahi mau palena.ʻOiai he pilina kiʻekiʻe ka RIN me ka maikaʻi o ka ʻikepili hoʻokolohua NGS, ʻaʻole kūpono ia no nā laʻana FFPE.Ua mālama ʻia nā laʻana FFPE no ka manawa lōʻihi, a he haʻahaʻa haʻahaʻa ka RNA i unuhi ʻia.Akā naʻe, ʻaʻole kēia manaʻo he pono ʻole ka ʻikepili kūpono o ka hoʻokolohua.No ka loiloi pono i ka maikaʻi o nā laʻana FFPE, pono mākou e hoʻohana i nā ana ʻē aʻe ma mua o RIN.Ma kahi o RIN, hiki i ka Agilent bioanalyzer ke helu i ka waiwai DV200 ma ke ʻano he loiloi loiloi o ka maikaʻi RNA.ʻO DV200 kahi ʻāpana e helu ana i ka hapa o nā ʻāpana ʻoi aku ka nui ma mua o 200 bp i kahi laʻana RNA.ʻO DV200 kahi hōʻailona ʻoi aku ka maikaʻi o ka laʻana FFPE ma mua o RIN.No ka RNA i unuhi ʻia e FFPE, he pilina kiʻekiʻe loa ia me ka helu o nā genes i hiki ke ʻike pono ʻia a me ka ʻokoʻa o nā genes [3].ʻOiai hiki i ka DV200 ke hoʻoponopono i nā hemahema i ka ʻike ʻana i ka maikaʻi o FFPE, ʻaʻole hiki i ka Agilent bioanalyzer ke hoʻopaʻa piha i nā pilikia maikaʻi i nā laʻana RNA, me ka loaʻa ʻana o nā inhibitors i loko o nā laʻana.Hiki i nā mea pale iā lākou iho ke hoʻopili i ka hoʻonui ʻana o nā hoʻokolohua i lalo a hoʻemi i ka nui o ka ʻikepili pono.I mea e ʻike ai inā he mea paʻa i loko o ka laʻana, hiki iā mākou ke hoʻohana i ke ʻano hana PCR fluorescent quantitative i wehewehe ʻia aʻe.

04 ʻO ka PCR nui o ka fluorescent manawa maoli

ʻAʻole hiki i ke ʻano PCR fluorescent quantitative maoli ke ʻike i nā inhibitors i loko o ka laʻana, akā no ka hōʻike pololei ʻana i ka maikaʻi o ka RNA i ka hāpana FFPE.Ke hoʻohālikelike ʻia me Agilent biological analyzers, ʻoi aku ka kaulana o nā mea hana fluorescence quantitative manawa maoli i nā laboratories biological nui ma muli o kā lākou noi ākea.No ka hoʻāʻo ʻana i ka maikaʻi o nā laʻana RNA, pono wale mākou e kūʻai a hoʻomākaukau i nā probes primer no nā genes kuhikuhi kūloko, e like me GUSB (Cat no. Hs00939627).Ma ka hoʻohana ʻana i kēia pūʻulu o nā primers, probes a me nā maʻamau (ka huina RNA o ka ʻike i ʻike ʻia) e hana i nā hoʻokolohua quantitative piha, hiki ke helu ʻia ka ʻāpana ʻāpana RNA kūpono e like me ke kūlana loiloi o ka maikaʻi RNA (Functional RNA Quantitation (FRQ) no ka pōkole).Ma kahi ho'āʻo NGS, ua ʻike mākou he pilina kiʻekiʻe loa ka FRQ o nā laʻana RNA me ka nui o ka ʻikepili kūpono.No nā laʻana a pau i ʻoi aku ma mua o 0.2ng/uL FRQ, hiki i ka 70% o nā heluhelu ke uhi pono i ke kaʻina kuhikuhi (Figure 4).

Kiʻi 4, ʻo ka waiwai FRQ i ʻike ʻia e ka fluorescence quantitative method he pilina kiʻekiʻe loa (R2>0.9) me ka ʻikepili kūpono i loaʻa i ka hoʻokolohua NGS.ʻO ka laina ʻulaʻula ka waiwai FRQ e like me 0.2 ng/uL (log10 = -0.7).【4】

Ma waho aʻe o ka pili ʻana i nā laʻana FFPE, hiki i ke kaʻina hana PCR nui manawa maoli ke nānā pono i nā mea hoʻopaneʻe i nā laʻana.Hiki iā mākou ke hoʻohui i ka hāpana e ʻike ʻia i loko o ka ʻōnaehana pane me ka Internal Positive Control (IPC) a me kāna Assay, a laila hana i ka quantification fluorescence e loaʻa ai ka waiwai Ct.Inā ua haʻalele ka waiwai Ct ma hope o ka waiwai Ct i ka hopena no-sample, hōʻike ia aia ka mea hoʻopaneʻe i loko o ka hāpana a keʻakeʻa i ka pono hoʻonui i ka hopena.

05 ʻO ke ʻano kumu hoʻolāʻau fluorescent Qubit

ʻO Qubit Fluorometer ka mea liʻiliʻi liʻiliʻi i hoʻohana pinepine ʻia no ka neʻe ʻana o ka waika nucleic a me ka ʻike maʻemaʻe, maʻalahi ke hana a loaʻa i loko o kēlā me kēia keʻena hana biology molecular.Heluhelu pololei ia i ka neʻe ʻana o ka ʻakika nucleic ma ka ʻike ʻana a me ka waika nucleic-binding fluorescent dye (Qubit detection reagent).He kiʻekiʻe ka naʻau a me ka kikoʻī o Qubit, a hiki ke hoʻohālikelike pololei i ka RNA a hiki i ka pg / µL ka nui.Ma waho aʻe o ka hiki ke ʻike pono i ka helu nucleic acid concentration, hiki i ka Thermo Fisher hou ke kumu hoʻohālike hou, Qubit 4.0, hiki ke ʻike i ka pono o RNA.ʻIke ʻo Qubit 4.0's RNA detection system (RNA IQ Assay) i ka pono o ka RNA ma ka ʻike like ʻana i ʻelua mau mea hoʻonaninani fluorescent.Hiki i kēia mau mea ʻoluʻolu ʻelua ke hoʻopaʻa i nā ʻāpana nui a me nā ʻāpana liʻiliʻi o RNA.Hōʻike kēia mau mea ʻoluʻolu ʻelua i ka hapa o nā ʻāpana nui o ka RNA i loko o ka hāpana, a mai kēia mea hiki ke helu ʻia ka waiwai IQ (Integrity and Quality) e hōʻike ana i ka maikaʻi RNA.Pili ka waiwai IQ i nā laʻana FFPE a me ka non-FFPE, a he mana nui kona i ka maikaʻi o ke kaʻina hana.Ke lawe nei i nā hoʻokolohua NGS ma ke ʻano he laʻana, ma nā hoʻokolohua hoʻokolohua RNA-Seq i hana ʻia ma ka platform Ion torrent™, ʻo ka hapa nui o nā laʻana me nā waiwai IQ i ʻoi aku ma mua o 4 he 50% ka heluhelu pono (Figure 5).Ke hoʻohālikelike ʻia me nā ʻano ʻike i ʻōlelo ʻia ma luna, ʻaʻole ʻoi aku ka maʻalahi o ka hana ʻana a Qubit IQ Assay a liʻiliʻi ka manawa (i loko o ʻelima mau minuke), akā he pilina nui nō hoʻi ma waena o ke ana ʻana i ka waiwai IQ a me ka maikaʻi o ka ʻikepili o nā hoʻokolohua lalo.

Kiʻi 5, aia kahi pilina nui ma waena o ka waiwai Qubit RNA IQ a me nā heluhelu palapala ʻia o RNA-Seq.【5】

Ma o ka hoʻolauna ʻana ma luna, ke manaʻoʻiʻo nei au ua lawa ka ʻike o kēlā me kēia kanaka i nā ʻano hana hoʻomalu maikaʻi RNA like ʻole.Ma ka hana, hiki iā ʻoe ke kohoke ala kūpono e like me ke ʻano hāpana a me nā mea hana e kū nei.Ma ka hoʻomalu wale ʻana i ka maikaʻi o ka RNA maikaʻi hiki iā mākou ke pale i ka hāʻule ʻole o nā hoʻokolohua ma hope mai ka maikaʻi ʻole o ka laʻana, no laila e mālama ai i ka manawa makamae, ka ikehu a me ke kumukūʻai.

Nā huahana kuhikuhi:

Huina Hoʻokaʻawale RNA Holoholona

Kelepona Huina RNA Isolation Kit

mau kuhikuhi

【1】Schroeder, A., Mueller, O., Stocker, S. et al.ʻO ka RIN: he helu kūpaʻa RNA no ka hāʻawi ʻana i nā waiwai kūpaʻa i nā ana RNA.BMC Molecular Biol 7, 3 (2006).https:// doi .org/10.1186/1471-21 99-7-3

【2】 Oncomine Human Immune Repertoire Guide User (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, Hoʻonui ʻia nā anana maikaʻi no ka loiloi ʻana i ka RNA i loaʻa mai ka Archival Formalin-Fixed Paraffin-Embedded Tissue Samples, Toxicological Sciences, Volume 170, Issue 3, Volume 170, Issue 3https://doi.org/10.1093/toxsci/