ʻO ka sterilization o nā ʻōlelo aʻoaʻo pipette a me nā paipu EP, etc.

1. E hoʻomākaukau i ka 0.1% (hoʻokahi tausani) DEPC (meaʻawaʻawa loa) me ka wai deionized, e hoʻohana pono ia i loko o kahi pahu hau, a mālama iā ia ma 4 ° C mai ka mālamalama;

ʻO ka wai DEPC ka wai maʻemaʻe i mālama ʻia me DEPC a sterilized e ka wela kiʻekiʻe a me ke kaomi kiʻekiʻe.Ua hoʻāʻo ʻia ʻaʻole i loaʻa iā RNase, DNase a me ka proteinase.

2. E kau i ka piko o ka pipette a me ka paipu EP i loko o ka 0.1% DEPC, a e hōʻoia i ka piha ʻana o ka piko o ka pipette a me ka paipu EP me 0.1% DEP.

3. Pale i ka malamalama, e ku, i ka po (12-24h)

4. ʻAʻole pono ka pahu i loko o ka piko a me ka paipu EP i DEPC.Ma hope o ka wehe ʻana i ka wai DEPC ma ka piko a i ʻole ka paipu EP, e hoʻopaʻa ʻia a ʻōwili ʻia.

5. 121 degere Celsius, 30 min

6. 180 degere Celsius, maloʻo no kekahi mau hola (ma kahi o 3 mau hola)

Nānā: a.E hoʻohana i nā mīkina lima latex a me nā maka i ka wā e lawelawe ai iā DEPC!b, a i ʻole ka DEPC sterilization, 130 ℃, 90min autoclave (nui nā laboratories kiʻekiʻe wela sterilization ʻelua)

Nā noʻonoʻo ʻana i ka RNA extraction

ʻElua hanana nui o ka hāʻule kaʻawale ʻana o ka RNA tissue

ʻO ka hoʻohaʻahaʻa RNA a me nā koena o nā haumia i loko o nā kiko,e pili ana i ka hoʻohaʻahaʻa ʻana, e nānā mua kākou i ke kumu ʻaʻole hiki ke hoʻohaʻahaʻa ʻia ka RNA i unuhi ʻia mai nā cell moʻomeheu.Loaʻa nā mea hoʻopaneʻe RNA i kēia manawa i nā ʻāpana e kāohi wikiwiki i ka RNase.E hoʻohui i ka lysate i nā keʻena moʻomeheu, a hui wale ia, hiki ke hui maikaʻi ʻia nā cell a pau me ka lysate, a ua lysed loa nā cell.Ma hope o ka hoʻokuʻu ʻia ʻana o nā cell, nā mea hana i loko o ka lysate e kāohi koke i ka RNase intracellular, no laila e paʻa mau ka RNA.ʻO ia hoʻi, no ka mea, ua maʻalahi a hoʻopili pono ʻia nā cell moʻomeheu me ka lysate, ʻaʻole maʻalahi ko lākou RNA;ma kekahiʻaoʻao, hiki ke hoʻohaʻahaʻaʻia ka RNA i loko o ke kino no ka meaʻaʻole maʻalahi nā pūnaewele i loko o ke kino e hoʻopili koke i ka lysate.ma muli o ka pili pono.No laila,Ke manaʻo nei aia kahi ala e hoʻohuli ai i ka ʻiʻo i hoʻokahi keena me ka pale ʻana i ka hana RNA, hiki ke hoʻopau piha ʻia ka pilikia o ka degradation.

ʻO ka wili nitrogen wai ka mea ʻoi loa o ia ʻano hana.Eia naʻe, pilikia loa ke ʻano o ka wili nitrogen wai, ʻoi aku ka nui o ka helu o nā laʻana.Hāʻawi kēia i kahi mea maikaʻi loa: ka homogenizer.ʻO kahomogenizerʻAʻole noʻonoʻo ke ala i ka nīnau pehea e pale ʻia ai ka hana RNase ma mua o ka hoʻopili ʻia ʻana o nā cell me ka lysate, akā ke pule nei i ʻoi aku ka wikiwiki o ka hoʻopau ʻana o ka ʻiʻo ma mua o ka nui o ka intracellular RNase e hoʻohaʻahaʻa ai i ka RN.

ʻOi aku ka maikaʻi o ka hopena o ka homogenizer uila,a ʻilihune ka hopena o ka homogenizer aniani, akā ma ka laulā, ʻaʻole hiki i ke ʻano homogenizer ke pale i ka hanana degradation.No laila, inā hoʻohaʻahaʻa ʻia ka unuhi ʻana, pono e hoʻohana ʻia ka homogenizer uila mua no ka wili ʻana me ka nitrogen wai;pono e hoʻololi i ka homogenizer aniani mua i homogenizer uila a i ʻole wili pololei ʻia me ka wai nitrogen.ʻO ka pilikia ma kahi o 100% hiki ke hiki.e hoʻoholo.

ʻOi aku nā kumu like ʻole o ke koena haumia e pili ana i nā hoʻokolohua aʻe ma mua o ka hoʻohaʻahaʻa ʻana, a ʻokoʻa nā hopena.I ka hopena,inā loaʻa ka hoʻohaʻahaʻa a i ʻole ke koena o nā haumia i loko o ka ʻiʻo, pono e hoʻomaikaʻi ʻia ke ʻano o ka unuhi ʻana/regent no ka mea hoʻokolohua kikoʻī.ʻAʻole pono ʻoe e hoʻohana i kāu mau hiʻohiʻona makamae no ka hoʻonui ʻana: hiki iā ʻoe ke kūʻai i kekahi mau holoholona liʻiliʻi e like me ka iʻa/moa mai ka mākeke, e lawe i ka ʻāpana kūpono o ka mea no ka unuhi ʻana i ka RNA, a ʻo kekahi ʻāpana no ka unuhi ʻana i ka protein - wili me ka waha, ka ʻōpū a me ka ʻōpū.

Hoʻohana ʻia ka RNA pahuhopu o ka RNA i unuhi ʻia no nā hoʻokolohua like ʻole, a he ʻokoʻa kona mau koi

Pono ke kūkulu hale waihona cDNA i ka pono o ka RNA me ka ʻole o nā koena o ka enzyme reaction inhibitors;Pono ka ʻĀkau i ka pono o ka RNA kiʻekiʻe a me nā koi haʻahaʻa no nā koena o ka enzyme reaction inhibitors;ʻAʻole koi ʻo RT-PCR i ka pono RNA kiʻekiʻe loa,aka, keakea nei i na hana enzyme.Koʻikoʻi nā koi koena.Hoʻoholo ka mea hoʻokomo i ka hoʻopuka;i kēlā me kēia manawa ka pahuhopu e loaʻa ka RNA maʻemaʻe kiʻekiʻe loa, e lilo ia i ka poʻe a me ke kālā.

ʻOhi / Waihona o nā Laʻana

Nā mea e pili ana i ka hoʻohaʻahaʻa ʻana Ma hope o ka haʻalele ʻana o ka laʻana i ke kino ola/a i ʻole ka ulu mua ʻana, e hoʻomaka ana nā enzyme endogenous i loko o ka hāpana e hoʻohaʻahaʻa i ka RNA,a pili ka helu degradation i ka ʻike o nā enzyme endogenous a me ka mahana.ʻO ka mea maʻamau, ʻelua wale nō ala e pale ai i ka hana enzyme endogenous: hoʻohui koke i ka lysate a homogenize maikaʻi a me ka wikiwiki;ʻoki liʻiliʻi a hoʻokuʻu koke i ka wai nitrogen.Pono nā ala ʻelua i ka hana wikiwiki.He kūpono ka hope no nā laʻana a pau, ʻoiai ʻo ka mea mua e kūpono wale no nā ʻiʻo me nā haʻahaʻa haʻahaʻa o nā cell a me nā enzyme endogenous a maʻalahi hoʻi e homogenize.ʻO ke kikoʻī, ʻoi aku ka maikaʻi o ka maloʻo me ka nitrogen wai ma mua o ka hoʻomaka ʻana o ka ʻiʻo mea kanu, ate, thymus, pancreas, spleen, lolo, momona, ʻiʻo ʻiʻo, etc.

ʻO ka māhele a me ka homogenization o nā laʻana

Nā mea e pili ana i ka hoʻohaʻahaʻa a me ka hāʻawi ʻana i ka ʻāpana laʻanano ka homogenization piha, ʻo ia no ka hoʻokuʻu piha ʻana o RNA.Hiki ke hoʻohui pono ʻia nā kelepona me ka ʻole o ka haki ʻana.Hiki ke hoʻohui ʻia nā ʻupena ma hope o ka haki ʻana.Pono e wāwahi ʻia ka hū a me ka bacteria me nā enzyme e pili ana ma mua o ka hiki ke hoʻohui ʻia.ʻO nā kiko me ka haʻahaʻa endogenous enzyme content a me ka homogenization maʻalahi hiki ke ʻoki ʻia a homogenized i ka manawa hoʻokahi i ka lysate e kahi homogenizer;ʻiʻo mea kanu, ate, thymus, pancreas, spleen, lolo, momona, ʻiʻo ʻiʻo a me nā laʻana ʻē aʻe, He kiʻekiʻe lākou i nā enzyme endogenous a ʻaʻole maʻalahi homogenizedno laila, pono e hoʻokaʻawale ʻia ka hoʻopau ʻana o ka ʻiʻo a me ka homogenization.ʻO ke ala ʻoi loa ka hilinaʻi a ʻoi aku ka maikaʻi o ka fragmentation ʻo ka wili ʻana me ka nitrogen wai, a ʻo ke ala kūpono loa o ka homogenization ka hoʻohana ʻana i kahi homogenizer uila.ʻO kahi leka kūikawā e pili ana i ka wili me ka nitrogen wai: ʻaʻole pono e hoʻoheheʻe ʻia ka hāpana i ke kaʻina wili holoʻokoʻa, no ka mea, ʻoi aku ka hana o nā enzyme endogenous i ka wā maloʻo.

Ke koho o ka lysate

Hoʻopilikia i ka ʻoluʻolu o ka hana a me nā kumu o ke koena o nā mea haumia endogenous ʻO nā mea hoʻonā lysis maʻamau i hoʻohana ʻia e aneane hiki ke kāohi i ka hana a RNase.No laila, ʻo ke kumu nui o ke koho ʻana i kahi hopena lysis e noʻonoʻo i ka hui pū ʻana me ke ʻano hoʻomaʻemaʻe.Aia kekahi mea ʻokoʻa:Hoʻohana ʻia nā laʻana me nā ʻano enzyme endogenous kiʻekiʻe e hoʻohana i ka lysate me ka phenol e hoʻonui ai i ka hiki ke hoʻopau i nā enzyme endogenous.

Ke koho ʻana i ke ʻano hoʻomaʻemaʻe

Nā mea e pili ana i ke koena o nā haumia endogenous, ka wikiwiki o ka unuhi ʻana No nā laʻana maʻemaʻe e like me nā cell, hiki ke loaʻa nā hopena ʻoluʻolu me kahi ʻano hoʻomaʻemaʻe ma ka lima.Akā no nā laʻana ʻē aʻe he nui, ʻoi aku ka nui o nā haumia kiʻekiʻe e like me nā mea kanu, ate, bacteria, etc., ke koho ʻana i kahi ala hoʻomaʻemaʻe kūpono.ʻO ke ʻano hoʻomaʻemaʻe centrifugal kolamu ka wikiwiki o ka unuhi ʻana a hiki ke hoʻopau pono i nā mea haumia e pili ana i ka hopena enzymatic hope o RNA, akā he kumukūʻai (Hiki iā Foregene ke hāʻawi i nā pahu kūʻai kumukūʻai, kaomi i nā kikoʻī hou aku.maanei);me ka hoʻohana ʻana i nā ʻano hoʻomaʻemaʻe maʻamau, e like me ka LiCl precipitation, hiki ke loaʻa nā hopena ʻoluʻolu, akā lōʻihi ka manawa hana..

"ʻEkolu Hoʻopaʻi a me ʻEwalu Manaʻo" no ka RNA Extraction

Aʻo 1:E hoʻopau i ka haumia o nā enzyme exogenous.

Nānā 1:E hoʻokomo pono i nā maka a me nā mīkina lima.

Nānā 2:ʻO nā paipu centrifuge, nā poʻo kipi, nā lāʻau pipette, nā pahu electrophoresis, a me nā noho hoʻokolohua i komo i ka hoʻokolohua pono e hoʻopau pono ʻia.

Nānā 3:ʻO nā reagents / hoʻonā i komo i ka hoʻokolohua, ʻoi aku ka wai, pono ʻole RNase-noa.

Aʻo 2:Kāohi i ka hana o nā enzyme endogenous

Nānā 4:E koho i kahi ala homogenization kūpono.

Nānā 5:E koho i kahi lysate kūpono.

Nānā 6:E hoʻomalu i ka nui hoʻomaka o ka hāpana.

Aʻo 3:E wehewehe i kāu kumu hoʻoiho

Nānā 7:Me kekahi ʻōnaehana lysate e hoʻokokoke ana i ka nui o ka hoʻomaka ʻana o ka laʻana, hāʻule nui ka helu lanakila o ka unuhi.

Nānā 8:ʻO ka mea hoʻokele waiwai wale nō no ka hoʻopau ʻana i ka RNA kūleʻa he kūleʻa i nā hoʻokolohua ma hope, ʻaʻole hua.

ʻO nā kumu 10 kiʻekiʻe o ka hoʻohaumia RNase

1. ʻO nā manamana lima ke kumu mua o nā enzymes exogenous, no laila pono e hoʻokomo a hoʻololi pinepine ʻia nā mīkina lima.Eia kekahi, pono e ʻaʻahu ʻia nā masks, no ka mea, ʻo ka hanu kekahi kumu nui o nā enzymes.ʻO kahi pōmaikaʻi ʻē aʻe o ke komo ʻana i ka pale pale lima ʻo ia ka pale ʻana i ka mea hoʻokolohua.

2. ʻAʻole hiki ke hoʻopau ʻia ka RNase ma ka sterilization wale nō, no laila pono e mālama ʻia nā ʻōlelo aʻoaʻo pipette a me nā paipu centrifuge me DEPC, ʻoiai inā e hōʻailona ʻia e like me DEPC i mālama ʻia.ʻOi aku ka maikaʻi o ka hoʻohana ʻana i kahi pipette kumu kūikawā, holoi ʻia me kahi pōpō pulupulu waiʻona 75% ma mua o ka hoʻohana ʻana, ʻoi aku ke koʻokoʻo;Eia hou, e hoʻomanaʻo ʻaʻole e hoʻohana i kahi wehe poʻo.

3. Pono ka wai/buffer me ka ʻole o ka haumia RNase.

4. Ma ka li'ili'i loa, pono e holoi 'ia ka papa ho'ā'o me nā pōlele pulupulu wai'ona he 75%.

5.Endogenous RNase Loaʻa i nā ʻiʻo a pau nā enzyme endogenous, no laila ʻo ka hoʻomaloʻo wikiwiki ʻana o nā ʻiʻo me ka nitrogen wai ke ala maikaʻi loa e hōʻemi ai i ka pōʻino.He mea maʻalahi ke ʻano o ka mālama ʻana i ka nitrogen wai, akā ʻo ia wale nō ke ala no nā kiko me nā kiʻekiʻe o nā enzyme endogenous.

6. Nā laʻana RNA Loaʻa paha i nā huahana hoʻoheheʻe RNA nā meheu o ka haumia RNase.

Hoʻohana pinepine ʻia ʻo Rnase e hoʻohaʻahaʻa i ka RNA, a ʻo ke koena Rnase e hoʻoheheʻe ʻia me Proteinase K a unuhi ʻia e PCI.

8. Hoʻopaʻa RNA ʻOiai inā mālama ʻia ma ka wela haʻahaʻa, ʻo ka nui o ka RNase ka mea e hōʻemi ai i ka RNA.ʻO ka hopena maikaʻi loa no ka mālama ʻana i ka RNA no ka wā lōʻihi, ʻo ia ka paʻakai a me ka waiʻona, no ka mea, ke kāohi nei ka waiʻona i nā hana enzymatic a pau i nā haʻahaʻa haʻahaʻa.

9. Ke loa'a kēia mau iona i nā cations (Ca, Mg), ho'omehana i ka 80C no nā minuke 5 e ho'oka'awale i ka RNA, no laila, inā pono ka RNA i ka wela, pono e loa'a i ka solution ho'omalu ka mea chelating agent (1mM Sodium Citrate, pH 6.4).

10. Hiki ke ho'ohaumia 'ia e RNase nā enzyme i ho'ohana 'ia ma nā ho'okolohua ma hope.

10 Manaʻo kōkua no ka RNA Extraction

1: E pale koke i ka hana RNase.Hoʻopili koke ʻia nā laʻana ma hope o ka hōʻiliʻili ʻana, a ua hoʻopau ʻia ʻo RNase e ka hana wikiwiki i ka wā lysis.

2: E koho i ke ʻano unuhi kūpono no ka ʻiʻo me ka ribozyme kiʻekiʻe, a ʻoi aku ka maikaʻi o ka ʻiʻo adipose e hoʻohana i ke ʻano me ka phenol.

3: Pono ka maikaʻi o ka wānana ma Northern, pono ke kūkulu hale waihona cDNA i ka kūpaʻa kiʻekiʻe, a ʻaʻole pono ʻo RT-PCR a me RPA (Ribonuclease protection assay) i ka pono kiʻekiʻe.Pono ka RT-PCR i ka maʻemaʻe kiʻekiʻe (nā koena inhibitor enzyme).

4: ʻO ka homogenization holoʻokoʻa ke kī i ka hoʻomaikaʻi ʻana i ka hua a me ka hōʻemi ʻana i ka degradation.

5: E nānā i ka pono o ka RNA electrophoresis detection, 28S: 18S = 2: 1 kahi hōʻailona piha, 1: 1 ʻae ʻia no ka hapa nui o nā hoʻokolohua.

6: Ka wehe 'ana i ka DNA no ka RT-PCR, array analysis 'Oi aku ka maika'i o ka ho'ohana 'ana iā Dnase I no ka wehe 'ana i ka DNA.

7: E ho'ēmi i ka haumia o nā enzymes exogenous - ʻaʻole hiki ke lawe ʻia nā enzyme mai waho.

8: I ka hoʻokomo ʻana i ka waikawa nucleic haʻahaʻa, pono e hoʻohui ʻia kahi reagent co-precipitation.Akā no ka pale ʻana i ka co-precipitant i loaʻa nā enzymes a me DNA contamination.

9: E hoʻopau loa i ka RNA, inā pono, e wela ma 65C no 5 mau minuke.

ʻano mālama kūpono

Hiki ke mālama ʻia ma -20C no ka manawa pōkole, a ma -80C no ka manawa lōʻihi.ʻO ka hana mua i ka hoʻomaikaʻi ʻana i nā hua RNA ʻo ka ʻike ʻana i ka ʻokoʻa o ka ʻike RNA o nā ʻokoʻa like ʻole.ʻO ka nui kiʻekiʻe (2-4ug/mg) e like me ka ate, pancreas, heart, medium abundance (0.05-2ug/mg) e like me ka lolo, embryo, kidney, lung, thymus, ovary, haʻahaʻa haʻahaʻa (<0.05ug/mg) mg) e like me ka ʻōpū, iwi, momona.

1: Lyse cell e hoʻokuʻu iā RN - inā ʻaʻole i hoʻokuʻu ʻia ka RNA, e hoʻemi ʻia ka hua.ʻOi aku ka maikaʻi o ka homogenization uila ma mua o nā ʻano homogenization ʻē aʻe, akā pono paha e hui pū ʻia me nā ʻano hana ʻē aʻe, e like me ka wai ʻana o ka nitrogen mashing, enzymatic digestion (Lysozyme/Lyticase)

2: Hoʻonui i ke ʻano o ka unuhi.ʻO nā pilikia nui me nā kaʻina hana phenol ʻo ka stratification piha ʻole a me ka nalowale ʻana o ka RNA hapa (ʻaʻole hiki ke hoʻopau loa ʻia ka supernatant).ʻO ka stratification incomplete ma muli o ke kiʻekiʻe o ka nucleic acid a me ka protein content, hiki ke hoʻoholo ʻia ma ka hoʻonui ʻana i ka nui o ka lysate i hoʻohana ʻia a i ʻole ka hōʻemi ʻana i ka nui o ka laʻana.Hoʻohui ʻia kahi ʻanuʻu o ka unuhi ʻana o ka chloroform i ka ʻiʻo adipose.Hiki ke hōʻemi ʻia ka pohō o ka RNA ma muli o ka hoʻomaʻamaʻa ʻana a i ʻole ka wehe ʻana i ka papa organik a ukali ʻia e ka centrifugation.ʻO ka pilikia nui loa me ke kolamu centrifugation-based method is excess sample.

Nā Manaʻo Hoʻoheheʻe Kūlana

1. Hoʻomaʻemaʻe Phenol: E hoʻohui i ka nui like o 1: 1 Phenol / Chloroform a hui ikaika no 1-2 mau minuke.ʻO Centrifuge ma ka wikiwiki kiʻekiʻe no 2 mau minuke.E wehe pono i ka supernatant (80-90%).Mai hele i ka papa waena.Hiki ke hoʻohui ʻia ka nui like o ka hopena hopena i Phenol/Chloroform a wehe ʻia ka supernatant.Hiki ke hoʻohui pū ʻia nā supernatant ʻelua no ka ua nucleic acid e hoʻomaikaʻi i ka hua.Mai akahai loa i ka hui ana, a mai hoao e wehe i ka supernatant.

2. Holoi me 70-80% ethanol: I ka wā e holoi ʻia ai, pono e hoʻokuʻu ʻia ka waikawa nucleic e hōʻoia i ka holoi ʻia ʻana o ke koena paʻakai.I ka manawa like, ma hope koke o ka ninini ʻana i ka ethanol, e hoʻokaʻawale i ke koena ethanol me ka pipette.E hoʻoheheʻe ma hope o ke kū ʻana i ka lumi wela no 5-10 mau minuke.

11. Wehe i nā hui kūikawā

1. Kaʻiʻo fibrous: ʻO ke kī i ka unuhi ʻana o RNA mai ka ʻiʻo fibrous e like me ka puʻuwai / ʻiʻo iwi ʻo ia ke hoʻopau loa i ka ʻiʻo.He haʻahaʻa ka nui o kēia mau ʻiʻo, no laila he haʻahaʻa ka nui o ka RNA no kēlā me kēia paona o ke kino, a ʻoi aku ka maikaʻi o ka hoʻohana ʻana i ka nui hoʻomaka e like me ka hiki.E hōʻoia e wili i ka ʻiʻo ma lalo o nā kūlana hau.

2. Nā kiko me ka protein kiʻekiʻe/ka momona: kiʻekiʻe ka momona o ka lolo/mea kanu.Ma hope o ka unuhi ʻana o PCI, loaʻa i ka supernatant nā floccules keʻokeʻo.Pono e unuhi hou ʻia ka supernatant me ka chloroform.

3. Nā kiko me ka nui o ka nucleic acid/ribozyme content: ʻo ka spleen/thymus he kiʻekiʻe ka nucleic acid a me ka ribozyme content.ʻO ka wili ʻana i ka ʻiʻo ma lalo o nā kūlana maloʻo a ukali ʻia e ka homogenization wikiwiki hiki ke hoʻopau pono i nā ribozymes.Eia nō naʻe, inā ʻeleʻele loa ka lysate (ma muli o ka nui o ka nui o ka nucleic acid), ʻaʻole hiki i ka PCI extraction ke stratify maikaʻi;Hiki i ka hoʻohui ʻana i ka lysate ke hoʻoponopono i kēia pilikia.Hiki i nā unuhi PCI he nui ke wehe i ke koena DNA.Inā puka koke ka wai keʻokeʻo ma hope o ka hoʻohui ʻana i ka waiʻona, hōʻike ia i ka hoʻohaumia DNA.Hiki i ka hoʻokuʻu hou ʻana me ka PCI acidic ma hope o ka hoʻoheheʻe ʻana e hiki ke hoʻopau i ka haumia DNA.

4. Kaʻiʻo mea kanu: ʻoi aku ka paʻakikī o ka ʻiʻo mea kanu ma mua o ka ʻiʻo holoholona.ʻO ka mea maʻamau, ʻāʻī ʻia nā mea kanu ma lalo o nā kūlana nitrogen wai, no laila ʻaʻole maʻamau ka hoʻohaʻahaʻa ʻana o RNA e nā enzyme endogenous.Inā ʻaʻole i hoʻoholo ʻia ka pilikia hoʻohaʻahaʻa, ʻaneʻane hiki ke kumu ʻia e nā haumia i loko o ka hāpana.ʻO nā mea haumia i loko o nā mea kanu he nui e alakaʻi i nā koena, a ʻo ke kumu o ke koena ʻo ia ka mea ma muli o kēia mau haumia i kekahi mau mea like me ka RNA: hoʻoheheʻe ʻoe a hoʻoheheʻe wau, a ʻo ʻoe hoʻi a hoʻopili wau.Hoʻoholo kēia mau hiʻohiʻona he ikaika loa lākou i ka enzyme inhibitors.

I kēia manawa, hiki ke hoʻololi ʻia nā mea hoʻoheheʻe RNA pāʻoihana i kahi kokoke i nā ʻiʻo holoholona āpau me nā hoʻololi liʻiliʻi, akā he liʻiliʻi nā mea hoʻoheheʻe RNA pāʻoihana hiki ke kūpono no ka hapa nui o nā mea kanu.ʻO ka mea pōmaikaʻi, hiki iā Foregene ke hāʻawi kūikawāmea kanu RNA pahu pahu, aia iā mākouMea kanu Huina RNA Isolation kit, Mea kanu Huina RNA Isolation kit Plus.Hoʻolālā kūikawā ʻia ka mea hope no nā mea kanu me ka polysaccharide kiʻekiʻe a me ka polyphenol content.No ka unuhi ʻana o RNA, maikaʻi loa nā manaʻo mai nā mea hoʻohana lab.

12. ʻO ka hopena o ka hoʻoheheʻe ʻana a me ka hoʻoheheʻe ʻana ʻO ka hāpana paʻa paʻa paha ʻoi aku ka nui, a pono e ʻoki ʻia ma mua o ka hoʻohana ʻana no ka unuhi ʻana i ka RNA.Hiki ke heheʻe (he hapa paha) i ka wā e ʻoki ai.Pono paha e kaupaona ʻia nā laʻana i hoʻopaʻa ʻia ma mua o ka unuhi ʻia ʻana o RNA, a e hana maoli ʻia ka hoʻoheheʻe ʻana i kēia kaʻina hana.I kekahi manawa, hiki mai ka thawing o ka hāpana i ka wā o ke kaʻina wili naikokene wai;ai'ole ka hāpana pa'a i ho'okomo pono 'ia i ka lysate me ka wili 'ole o ka naikokene wai, a e hana maoli 'ia ka thawing ma mua o ka homogenization piha.Ua hōʻike ʻia nā hoʻokolohua ʻoi aku ka maʻalahi o ka ʻiʻo paʻa i ka hōʻino ʻana o RNA i ka wā e hoʻoheheʻe ʻia ma mua o ka ʻiʻo hou.ʻO ke kumu paha: ʻO ke kaʻina manuahi-thaw e hoʻopau i nā hale i loko o ke keʻena, e maʻalahi i nā enzyme endogenous ke komo pololei me ka RNA.

13. Hoʻoholo i ka maikaʻi RNA ʻO ka maʻamau, hoʻohana ʻia ka electrophoresis e hoʻoholo i ka pono o RNA, a hoʻohana ʻia ʻo A260/A280 e hoʻoponopono i ka maʻemaʻe o RNA.Ma ke kumumanaʻo, loaʻa i ka RNA intact ka ratio o 28S:18S = 2.7:1, a ʻo ka hapa nui o nā ʻikepili e hoʻokūpaʻa i ka ratio o 28S:18S = 2:1.ʻO ka mea ʻoiaʻiʻo, ʻaʻohe mea o ka RNA i unuhi ʻia mai nā laʻana ma waho o nā cell i loko o ka ratio 2: 1 (ua loaʻa kēia me ka Agilent Bioanalyzer).

Hoʻopili ʻia nā hopena electrophoresis o RNA e nā kumu he nui, ʻo ia hoʻi ke ʻano lua, nā kūlana electrophoresis, ka hāpana hāpana, ka degere o ka saturation e EB, a pēlā aku.Inā maopopo ka 28S ma 2kb a me ka 18S ma 0.9kb, a ʻo 28S: 18S > 1, hiki i ka pono ke hoʻokō i nā koi o ka hapa nui o nā hoʻokolohua ma hope.

ʻO A260/A280 kahi hōʻailona i hoʻokumu i ka huikau nui.ʻO ka mea mua, pono e wehewehe i ke ʻano kumu o kēia hōʻailona no nā waikawa nucleic: RNA maʻemaʻe, kona A260/280 = ma kahi o 2.0.ʻO RNA maʻemaʻe ka 'kumu' a ʻo A260/A280 = 2 ka 'hopena'.Ke hoʻohana nei nā mea a pau iā A260/A280 ma ke ʻano he 'kumu', me ka manaʻo "inā ʻo A260/A280 = 2, a laila maʻemaʻe ʻo RNA", e alakaʻi maoli i ka huikau.

Inā makemake ʻoe, hiki iā ʻoe ke hoʻohui i kahi reagent liʻiliʻi i hoʻohana pinepine ʻia i ka unuhi ʻana, e like me phenol, guanidine isothiocyanate, PEG, etc., i kāu hāpana RNA, a laila e ana i ka ratio A260/A280.ʻO ka mea ʻoiaʻiʻo, ʻo ka nui o nā reagents i hoʻohana ʻia no ka unuhi ʻana o RNA, a me nā mea haumia he nui i loko o ka laʻana, e komo a puni ka A260 a me A280, e pili ana iā A260/A280.

ʻO ke ala hoʻonaʻauao loa i kēia manawa, ʻo ia ka nānā ʻana i nā laʻana RNA ma ka laulā 200-300 nm.ʻO ka pihi o ka RNA maʻemaʻe nā hiʻohiʻona penei: ʻoluʻolu ka pihi, A230 a me A260 ʻelua mau helu inflection, A300 kokoke i 0, A260/A280 = ma kahi o 2.0, a me A260/A230 = ma kahi o 2.0.Inā ʻaʻole i loaʻa ka ʻikepili scan, pono e hoʻoholo ʻia ka ratio A260/A230, no ka mea, ʻoi aku ka maʻalahi o kēia ratio i ka lawe ʻana i nā haumia āpau e pili ana i ka hopena enzymatic.E noʻonoʻo i ka laina laina o ka hāmeʻa (0.1–0.5 no A260).

ʻElua mau mea kūpono ʻē aʻe: ʻo ka ratio ma kahi o 0.3 haʻahaʻa ke ana ʻia ʻo A260/A280 i ka wai;ʻoiai ʻo ka lakio i ana ʻia ma 10 mM EDTA ma kahi o 0.2 kiʻekiʻe ma mua o ka mea i ana ʻia ma 1 mM EDTA.

Nā huahana pili:

China Plant Total RNA Isolation Kit Manufacturer and Supplier |Foregene (foreivd.com)

Nā mea kūʻai aku a me nā hale hana RNA |Nā mea hana kaʻawale RNA RNA Kina (foreivd.com)

RNA kaʻawale ʻana - Foregene Co., Ltd. (foreivd.com)