Kararehe Tapeke RNA Isolation Kete Tapeke RNA Tangohanga me te purenga kete mo nga Tissue kararehe me te Pūtau
Whakaahuatanga kete:
He tere me te whai hua te tango i te RNA tapeke tino parakore me te kounga teitei mai i nga momo kopa kararehe.
Kaore he take ki te awangawanga mo te whakahekenga o te RNA.Ko te punaha katoa he RNase-Kore
He pai te tango i te DNA ma te whakamahi i te Tiwae Horoi DNA
Tangohia te DNA me te kore e taapiri i te DNAase
He ngawari—ka oti nga mahi katoa i te pāmahana rūma
Tere—ka oti te mahi i roto i te 30 meneti
Haumaru—kaore he reagent waro i whakamahia
Te parakore teitei—OD260/280≈1.8-2.1
Whakaahuatanga kete
50 Preps, 200 Preps
Ka whakamahia e tenei kete tetīwae miro me te tātaii whakawhanakehia e to maatau kamupene, ka taea te tango i te RNA katoa o te maa me te kounga teitei mai i nga momo kararehe kararehe me te tino pai.Ka whakaratohia e ia he Tiwae Whakapai DNA pai, ka taea te wehe me te whakauru i te DNA genomic mai i te supernatant me te kiko lysate, ngawari me te wa-whakaora;Ka taea e te Tiwae RNA-anake te here i te RNA ka taea te tukatuka i te wa kotahi me te tauira ahurei He maha nga tauira.
Ko te katoa o te punaha he RNase-Free, kia kore ai te RNA kua tangohia mai e whakaheke;Pūnaha Buffer RW1, Buffer RW2 Buffer washing system, kia kore ai te RNA i whiwhi i te pūmua, te DNA, te katote, me te parahanga pūhui waro.
Nga waahanga kete
| Kararehe Tapeke RNA Isolation Kit | ||
| Nga waahanga kete | RE-03011 | RE-03014 |
| 50 T | 200 T | |
| Pupuri RL1* | 25ml | 100ml |
| Kaipupuri RL2 | 15ml | 60ml |
| Pupuri RW1* | 25ml | 100ml |
| Kaipupuri RW2 | 24ml | 96ml |
| RNase-Free ddH2O | 10ml | 40ml |
| RNA-anake Tīwae | 50 | 200 |
| Tiwae Whakapai DNA | 50 | 200 |
| Pukapuka Tohutohu | 1 wahi | 1 wahi |
Nga korero hua
| Whakahōputu | Tīwae hurihuri | Wae purenga | Tuhinga o mua, reagent |
| Rereke | 1-24 tauira | Te wa mo ia whakaritenga | ~30 min (24 tauira) |
| Pūwhakaaro | Puka tepu | Te wehenga Pyrolysis | Te wehenga centrifugal |
| Tauira | Te kiko kararehe;pūtau | Te rahinga tauira | Tiu: 10-20 mg;Pūtau:(1-5)×106 |
| rōrahi elution | 50-200 μL | Rōrahi utanga mōrahi | 850 μL |
Nga waahanga me nga painga
■ Kaua e manukanuka ki te whakahekenga o te RNA;he RNase-Free te punaha katoa
■ Kia pai te tango i te Tiwae Horoi-ADN ma te whakamahi i te DNA
■ Tangohia te DNA me te kore e taapiri i te DNase
■ Maamaa-ka oti nga mahi katoa i te pāmahana rūma
■ Ka taea te mahi tere i roto i te 30 meneti
■ Haumaru-kare he reagent waro e hiahiatia ana
■ Te parakore teitei -OD260/280≈1.8-2.1
Te tono kete
He pai mo te tangohanga me te purenga o te RNA katoa mai i nga momo momo kopa kararehe hou, tio ranei, nga pūtau whakatipu.
Tawhā hua
■ Nga tono whakararo: te whenu tuatahi cDNA synthesis, RT-PCR, molecular cloning, Northern Blot, etc.
■ Tauira: kopa kararehe, pūtau whakatipu
■ Inenga: Tissues 10-20mg, Pūtau(2-5)×106
■ Te kaha herenga DNA morahi o te pou purenga: 80 μg
■ Rōrahi Elution: 50-200 μl
Hoahoa
Kararehe Tapeke RNA Isolation Kit i rongoatia 20mg
Ko nga tauira kiore hou, tangohia te 5% RNA katoa kua purea 1% agar
Glycogel electrophoresis
1: Spleen 2: Tākihi
3: ate 4: Ngakau
Te rokiroki me te oranga o te papa
Ka taea te penapena i te kete mo te 24 marama ki te pāmahana rūma (15-25 ℃) 2-8 ℃ ranei mo te wa roa.Ka taea te penapena Buffer RL1 ki te 4 ℃ mo te 1 marama i muri i te taapiri i te β-mercapptoethanol (he kowhiri).
Nga tuhinga kua whakahuahia
1.Mena:18.808:Zheng, Q., Qin, F., Luo, R., et al.MRNA-Utaina Lipid-rite Nanoparticles mo te Whakatika turanga ate Na roto i te arotautanga o Central Composite Hoahoa.Adv.Mahi.Mater.2021, 31, 2011068.doi:10.1002/adfm.202011068.
2.Mena:18.187:He X, Hong W, Yang J, et al.Ko te apoptosis mahorahora o nga pūtau i roto i te whakarite i nga pūtau kakau rongoa ka whai hua immunomodulatory na roto i te tukunga o te phosphatidylserine.Tohu Whakawhiti Whainga Whai.2021 Hūrae 14;6(1):270.doi: 10.1038/s41392-021-00688-z.
3.Mena:17.97:Dai Z, Liu H, Liao J, et al.Ko te whakarereketanga o te N7-Methylguanosine tRNA e whakanui ana i te whakamaoritanga mRNA oncogenic me te whakatairanga i te ahunga whakamua o te cholangiocarcinoma intrahepatic.Pūtau Mol.2021 Hūrae 29:S1097-2765(21)00555-4.doi: 10.1016/j.molcel.2021.07.003.
4.Mena:9.225:Cao X, Shu Y, Chen Y, et al.Ko te Mettl14-Mediated m6A Whakarereketanga Ka Whakapaihia te Whakahoutanga Ate ma te pupuri i te Endoplasmic Reticulum Homeostasis.Pūtau Mol Gastroenterol Hepatol.2021;12(2):633-651.doi: 10.1016/j.jcmgh.2021.04.001.
Ko nga kete wehe RNA mo etahi atu puna tauirakei te wātea:
Pūtau, tipu, viral, toto, etc.
Kaore te RNA e tangohia, he iti ranei nga hua RNA
He maha nga momo ahuatanga e pa ana ki te pai o te whakaora, penei i: tauira kiko RNA ihirangi, tikanga mahi, rōrahi elution, etc.
1. I mahia te kaukau tio, cryogenic (4 °C) centrifugation i te wa e mahi ana.
Taunakitanga: Mahi i te pāmahana rūma (15-25 ° C) puta noa i te tukanga katoa, kaua e kaukau i te hukapapa me te centrifuge i te pāmahana iti.
2. He pai te tiaki tauira, he nui rawa te waa rokiroki tauira.
Taunakitanga: Whakaorangia nga tauira ki te -80 °C, whakatio ranei i roto i te hauota wai me te karo i te whakamahi whakatio-rewa;ngana ki te whakamahi i te kiko hou, i nga pūtau ahurea ranei mo te tangohanga RNA.
3. He iti rawa te tauira lysis.
Taunakitanga: I te wa e whakakotahi ana i te kiko, me whakarite kia rite te kiko o te kiko me te wehea nga pūtau kiko hei whakamarama i te tukunga o te RNA.
4. Karekau i tika te taapiri i te eluent.
Taunakitanga: Whakaū ko RNase-Free ddH2Ka taapirihia te O ki te taha ki waenganui o te kiriuhi pou purenga.
5. Ko te rōrahi tika o te waihā tino kore i tāpirihia ki te Buffer RL2, te Buffer RW2 ranei.
Taunakitanga: Whaia nga tohutohu, taapirihia te rōrahi tika o te waihā pūmau ki te Buffer RL2 me te Buffer RW2 ka uru pai i mua i te whakamahi i te kete.
6. Ko te inenga tauira o te kopa kaore e tika.
Taunakitanga: Whakamahia te 10-20 mg o te kiko (1-5) × 10 ranei6nga pūtau mo te 500 μl buffer RL1, na te nui o te whakamahi kiko ka taea te whakaheke i te tangohanga RNA.
7. Ko te rōrahi elution hē, te hauhā kore rānei.
Taunakitanga: Ko 50-200 μl te rōrahi elution o te tīwae pure;Mena kaore i te pai te paanga o te whakamaarama, ka tūtohu kia whakaroahia te wa tuunga pāmahana rūma i muri i te taapiri i te RNase-Free ddH kua whakamahana.2O, hei tauira mo te 5-10 min.
8.Ko te tīwae purenga he toenga waihā i muri i te horoinga Buffer RW2.
Taunakitanga: Mena he toenga waihano i muri i te horoinga o te Buffer RW2, te centrifugation ngongo kau mo te 1min, ka taea te whakanui ake i te wa mo te mahi centrifugation ngongo kau ki te 2min, ka taea ranei te whakanoho i te pou purea ki te pāmahana rūma mo te 5 min ki te tango tika i te toenga waiwaro.
Kua whakahekehia te RNA kua purea
Ko te kounga o te RNA kua purea e pa ana ki nga ahuatanga penei i te tiaki i te tauira, te poke RNase, me te raweke, me era atu.
1. Ko nga tauira o te kopa kaore i te pupuri i te waa.
Taunakitanga: Ki te kore e whakamahia nga tauira kiko, nga pūtau ranei i te wa tika i muri i te kohi, ka tiakina tonutia i te -80 °C, hauota wai ranei.Hei tango i te RNA, whakamahia he kiko hou, he tauira pūtau ranei ka taea.
2. Te whakatio-whakarewa o nga tauira kiko.
Taunakitanga: I te wa e penapena ana i nga tauira kiko, he pai ake kia tapahia ki nga wahi iti mo te tiaki, ka tango i tetahi o nga waahanga ina whakamahia kia kore ai e rewa-rewa te tauira me te paheketanga o te RNA.
3. Ka whakauruhia te RNase, kaore ranei e mau i nga karapu tuku, he kanohi kanohi, me etahi atu i te wa e mahi ana.
Taunakitanga: He pai rawa atu nga whakamatautau tangohanga RNA i roto i nga ruma raweke RNA motuhake, ka whakawāteahia te teepu i mua i te whakamatautau.
Whakamaua nga karapu tuku me nga kanohi kanohi i te wa o te whakamatautau hei whakaiti i te paheketanga o te RNA na te whakaurunga o te RNase.
4. Kua poke nga reagents ki te RNase i te wa e whakamahia ana.
Taunakitanga: Whakakapihia ki te Kete Motuhake RNA Tapeke Kararehe hou mo nga whakamatautau e pa ana.
5. Ko nga ngongo centrifuge, nga tohutohu, me era atu e whakamahia ana i roto i te whawhe RNA kua poke ki te RNase.
Taunakitanga: Whakauhia ko nga ngongo centrifuge, nga tohutohu, nga pipettes, me etahi atu e whakamahia ana i te tangohanga RNA he RNase-Kore katoa.
Ko te RNA kua purea ka pa ki nga whakamatautau o raro
Ko te RNA kua purea e te pou purenga, mena he nui rawa nga katote tote, he nui rawa te ihirangi pūmua ka pa ki te whakamatautau whakararo, penei: te whakamuri whakamuri,Northern Blot et al.
1. He toenga katote tote kei te RNA kua whakahiahia.
Taunakitanga: Whakapumautia kua taapirihia te rōrahi tika o te waihā ki te Buffer RW2 ka mahia kia 2 nga horoinga o te pou purenga i te tere o te hau i tohuhia mo te mahi;ki te mea he toenga katote tote, waiho te pou purenga ki te Buffer RW2 mo te 5 meneti i te pāmahana rūma me te mahi centrifugation ki te whakanui i te tangohanga o te poke tote.
2. Te toenga o te waihano i roto i te RNA elutioned.
Taunakitanga: Whakapumauhia i muri i te horoinga o te RW2, mahia te mahi centrifugation ngongo kau i te tere centrifugation e tohuhia ana mo te mahi, whakanuia te wa o te mahi centrifugation ngongo kau ki te 2 min mena kei te noho tonu te toenga waihano, waiho ranei i te pāmahana rūma mo te 5 min i muri i te centrifugation ngongo kau hei whakanui ake i te tangohanga o te toenga waihano.
