Ikhithi Yokuhlukanisa Yezilwane Ingqikithi Ye-RNA Ingqikithi Yekhithi Ye-RNA Yokukhipha Nezihlanzo Zezicubu Zezilwane Neseli
Incazelo yekhithi:
Khipha ngokushesha nangempumelelo ukuhlanzeka okuphezulu nekhwalithi ephezulu ingqikithi ye-RNA ezicutshini zezilwane ezahlukahlukene.
Asikho isidingo sokukhathazeka ngokuwohloka kwe-RNA.Lonke uhlelo alunayo i-RNase-Free
Khipha ngempumelelo i-DNA usebenzisa i-DNA-Cleaning Column
Susa i-DNA ngaphandle kokwengeza i-DNase
Kulula—yonke imisebenzi iqedwa ezingeni lokushisa legumbi
Ukusheshisa—ukusebenza kungaqedwa ngemizuzu engu-30
Iphephile—ayikho i-organic reagent esetshenzisiwe
Ukuhlanzeka okuphezulu—OD260/280≈1.8-2.1
Kits Izincazelo
50 Preps, 200 Preps
Le kit isebenzisa i-spin ikholomu kanye nefomulaithuthukiswe inkampani yethu, engakwazi ukukhipha i-RNA ehlanzekile nesezingeni eliphezulu ezicutshini zezilwane ezihlukahlukene ngokusebenza kahle okuphezulu.Ihlinzeka ngekholomu ephumelelayo ye-DNA-Cleaning, engakwazi ukuhlukanisa kalula futhi ikhangise i-DNA ye-genomic kusuka ku-supernatant kanye ne-tissue lysate, elula futhi elondoloza isikhathi;Ikholomu ye-RNA kuphela ingahlanganisa ngempumelelo i-RNA futhi ingacutshungulwa kanyekanye ngefomula eyingqayizivele Amasampuli amaningi.
Lonke uhlelo luyi-RNase-Free, ukuze i-RNA ekhishiwe ingonakalisiwe;I-Buffer RW1, i-Buffer RW2 isistimu yokugeza ibhafa, ukuze i-RNA etholiwe ingabi nawo amaprotheni, i-DNA, i-ion, kanye nokungcoliswa kwenhlanganisela yezinto eziphilayo.
Izingxenye zekhithi
| Ikhithi Yokuhlukanisa Yezilwane Ephelele ye-RNA | ||
| Izingxenye zekhithi | RE-03011 | RE-03014 |
| 50 T | 200 T | |
| I-Buffer RL1* | 25ml | 100ml |
| Isilondolozi RL2 | 15ml | 60ml |
| Isilondolozi RW1* | 25ml | 100ml |
| Isilondolozi RW2 | 24ml | 96ml |
| RNase-Free ddH2O | 10ml | 40ml |
| Ikholomu ye-RNA kuphela | 50 | 200 |
| Ikholomu Yokuhlanza I-DNA | 50 | 200 |
| Incwadi Yeziqondiso | 1 isiqephu | 1 isiqephu |
Ulwazi lomkhiqizo
| Ifomethi | Spin ikholomu | Ingxenye yokuhlanza | Ikholomu ye-Foregene, i-reagent |
| I-Flux | 1-24 amasampula | Isikhathi sokulungiselela | ~30 amaminithi (24 amasampula) |
| I-Centrifuge | I-centrifuge yedeski | Ukuhlukaniswa kwe-pyrolysis | Ukuhlukaniswa kwe-Centrifugal |
| Isampula | Izicubu zezilwane;iseli | Inani lamasampuli | Izicubu: 10-20 mg;Iseli:(1-5)×106 |
| Ivolumu ye-Elution | 50-200 μL | Ivolumu yokulayisha ephezulu | 850 μL |
Izici&izinzuzo
■ Asikho isidingo sokukhathazeka ngokuwohloka kwe-RNA;lonke uhlelo luyi-RNase-Free
■ Khipha ngempumelelo i-DNA usebenzisa i-DNA-Cleaning Column
■ Khipha i-DNA ngaphandle kokwengeza i-DNase
■ Kulula-yonke imisebenzi iqedwa ekamelweni lokushisa
■ Ukusebenza okusheshayo kungaqedwa ngemizuzu engama-30
■ I-Safe-akukho reagent ephilayo edingekayo
■ Ukuhlanzeka okuphezulu -OD260/280≈1.8-2.1
Isicelo sekhithi
Ilungele ukukhishwa nokuhlanzwa kwe-RNA ephelele ezinhlobonhlobo zezicubu zezilwane ezintsha noma eziqandisiwe noma amaseli akhulisiwe.
Imingcele yomkhiqizo
■ Izinhlelo zokusebenza eziwela phansi: ukuhlanganiswa kwe-cDNA ye-first-strand, i-RT-PCR, i-molecular cloning, i-Northern Blot, njll.
■ Amasampula: izicubu zezilwane, amaseli akhulisiwe
■ Umthamo: Izicubu 10-20mg, Amaseli(2-5)×106
■ Umthamo omkhulu wokubopha we-DNA wekholomu yokuhlanza: 80 μg
■ Ivolumu ye-Elution: 50-200 μl
Umdwebo
I-Animal Total RNA Isolation Kit iphathwe ama-20mg
Amasampula egundane amasha, thatha i-RNA ephelele engu-5% 1% ye-agar
I-Glycogel electrophoresis
1: Ubende 2: Izinso
3: Isibindi 4: Inhliziyo
Isitoreji nempilo yeshelufu
Ikhithi ingagcinwa izinyanga ezingama-24 ekamelweni lokushisa (15-25 ℃) noma 2–8 ℃ isikhathi eside.I-Buffer RL1 ingagcinwa ku-4 ℃ inyanga engu-1 ngemva kokwengeza i-β- mercaptoethanol (uma uthanda).
Izihloko ezicashuniwe
1.IF:18.808:Zheng, Q., Qin, F., Luo, R., et al.I-mRNA-Layishwa I-Lipid-Like Nanoparticles Yokuhlelwa Kwesisekelo Sesibindi Ngokuthuthukiswa Komklamo Oyinhlanganisela Emaphakathi.Adv.Umsebenzi.UMater.2021, 31, 2011068.doi:10.1002/adfm.202011068.
2.IF:18.187:He X, Hong W, Yang J, et al.I-apoptosis ezenzakalelayo yamaseli ekulungiseleleni i-stem cell therapy isebenzisa imiphumela yokuzivikela ngokukhululwa kwe-phosphatidylserine.I-Signal Transduct Target Ther.2021 Jul 14;6(1):270.doi: 10.1038/s41392-021-00688-z.
3.IF:17.97:Dai Z, Liu H, Liao J, et al.Ukuguqulwa kwe-N7-Methylguanosine tRNA kuthuthukisa ukuhumusha kwe-oncogenic mRNA futhi kukhuthaze ukuqhubeka kwe-intrahepatic cholangiocarcinoma.Mol Cell.2021 Jul 29:S1097-2765(21)00555-4.doi: 10.1016/j.molcel.2021.07.003.
4.IF:9.225:Cao X, Shu Y, Chen Y, et al.Ukuguqulwa kwe-M6A kwe-Mettl14-Mediated Kusiza Ukuvuselelwa Kwesibindi Ngokugcina I-Endoplasmic Reticulum Homeostasis.Cell Mol Gastroenterol Hepatol.2021;12(2):633-651.doi: 10.1016/j.jcmgh.2021.04.001.
I-RNA isoaltion kits ye eminye imithombo yesampulaayatholakala:
Iseli, isitshalo, igciwane, igazi, njll.
I-RNA ayikhishiwe noma isivuno se-RNA siphansi
Kuvame ukuba nezinto ezihlukahlukene ezithinta ukusebenza kahle kokululama, njengalezi: okuqukethwe kwesampula yezicubu ze-RNA, indlela yokusebenza, ivolumu ye-elution, njll.
1. Ukugeza kweqhwa noma i-cryogenic (4 °C) i-centrifugation yenziwa ngesikhathi sokusebenza.
Okutuswayo: Sebenzisa ekamelweni lokushisa (15-25 ° C) kuyo yonke inqubo, ungabhavi eqhweni kanye ne-centrifuge emazingeni okushisa aphansi.
2. Ukulondolozwa kwesampula okungalungile noma isikhathi sokugcina isampula eseqile.
Okutuswayo: Gcina amasampula ku--80 °C noma ufrize ku-nitrogen ewuketshezi futhi ugweme ukusetshenziswa okuphindaphindiwe kokuqhwaza;zama ukusebenzisa izicubu ezintsha noma amaseli akhulisiwe ukuze kukhishwe i-RNA.
3. Isampula yesampula enganele.
Okutuswayo: Uma wenza izicubu zibe homogenizing, qinisekisa ukuthi izicubu zihlanganiswe ngendlela eyanele nokuthi amaseli ezicubu ahlukaniswe ngokwanele ukuchaza ukukhululwa kwe-RNA.
4. I-eluent ayengezwanga ngendlela efanele.
Okunconyiwe: Qinisekisa ukuthi i-ddH ye-RNase-Free2U-O wengezwa ngokudonsela phansi maphakathi nolwelwesi lwekholomu yokuhlanza.
5. Ivolumu elungile ye-ethanol ephelele ayizange yengezwe ku-Buffer RL2 noma ku-Buffer RW2.
Isincomo: Landela imiyalelo, engeza ivolumu efanele ye-ethanol ephelele ku-Buffer RL2 kanye ne-Buffer RW2 bese uxuba kahle ngaphambi kokusebenzisa ikhithi.
6. Umthamo wesampula wezicubu awufanelekile.
Isincomo: Sebenzisa i-10-20 mg yezicubu noma (1-5) × 106amaseli nge-500 μl buffer RL1, njengoba ukusetshenziswa kwezicubu ngokweqile kungaholela ekukhishweni kwe-RNA okuncishisiwe.
7. Ivolumu ye-elution engafanele noma ukungezwani okuphelele.
Okutuswayo: Ivolumu ye-elution yekholomu yokuhlanza ingu-50-200 μl;uma umphumela we-elution ungagculisi, kuyanconywa ukuthi kunwetshwe isikhathi sokubeka izinga lokushisa legumbi ngemva kokwengeza i-ddH eshisiwe ye-RNase-Free2O, isb imizuzu emi-5-10.
8.Ikholomu yokuhlanza inensalela ye-ethanol ngemva kokugeza kwe-Buffer RW2.
Okutuswayo: Uma kunensalela ye-ethanol ngemva kokugeza kwe-Buffer RW2, i-tube centrifugation engenalutho ye-1min, isikhathi sokusebenza kwe-tube centrifugation esingenalutho singanyuswa sibe yi-2min, noma ikholomu yokuhlanza ingabekwa ekamelweni lokushisa imizuzu emi-5 ukuze kukhishwe ngokwanele i-ethanol esele.
I-RNA ehlanziwe yehlisiwe
Ikhwalithi ye-RNA ehlanzekile ihlobene nezici ezifana nokulondolozwa kwesampula, ukungcoliswa kwe-RNase, nokukhohlisa, njll.
1. Amasampula ezicubu awagcinwa ngesikhathi.
Okutuswayo: Uma amasampula ezicubu noma amaseli engasetshenziswa ngesikhathi esifanele ngemva kokuqoqwa, londoloza ngokushesha i-cryopreserve ku- -80 °C noma i-nitrogen ewuketshezi.Ukuze ukhiphe i-RNA, sebenzisa ithishu esanda kuthathwa noma isampula yeseli noma nini lapho kunokwenzeka.
2. Ukuqhwaza okuphindaphindiwe kwamasampula ezicubu.
Okutuswayo: Uma ugcina amasampula ezicubu, kuhle kakhulu ukuwasika abe izingcezu ezincane ukuze alondolozwe, bese ususa ingxenye eyodwa yezingcezu lapho uzisebenzisa ukugwema ukuncibilika okubandayo kwesampula kanye nokonakala kwe-RNA.
3. I-RNase yethulwa noma ingagqoki amagilavu alahlwayo, imaski, njll. phakathi nokuhlinzwa.
Okunconyiwe: Ukuhlolwa kokukhipha i-RNA kwenziwa kangcono kakhulu emagumbini okukhohlisa e-RNA ahlukene futhi ithebula liyasulwa ngaphambi kokuhlolwa.
Gqoka amagilavu alahlwayo kanye nemaski ngesikhathi sokuhlolwa ukuze unciphise ukucekelwa phansi kwe-RNA okubangelwa ukwethulwa kwe-RNase.
4. Ama-reagents angcoliswe yi-RNase ngesikhathi sokusetshenziswa.
Okunconyiwe: Faka esikhundleni Ikhithi Yokuhlukanisa Yezilwane Ephelele Yenani Le-RNA ukuze uthole ukuhlolwa okuhlobene.
5. Amashubhu e-centrifuge, amathiphu, njll. asetshenziswa ekukhohlisweni kwe-RNA angcoliswe yi-RNase.
Okutuswayo: Qinisekisa ukuthi amashubhu e-centrifuge, amathiphu, amapayipi, njll. asetshenziswa ekukhishweni kwe-RNA konke Akuna-RNAse.
I-RNA etholiwe ehlanziwe ithinta ukuhlola okwehla nomfula
I-RNA ehlanjululwe ngekholomu yokuhlanza, uma ama-ion kasawoti, okuqukethwe kwamaprotheni kukukhulu kakhulu kuzothinta isilingo esisezansi nomfula, njengokuthi: ukuloba okuhlanekezelwe,Northern Blot et al.
1. I-RNA ekhishiwe inezinsalela ze-ion kasawoti.
Okunconyiwe: Qinisekisa ukuthi ivolumu elungile ye-ethanol yengezwe ku-Buffer RW2 futhi wenze amakholomu okuhlanza ama-2 ageza ngesivinini esimaphakathi esikhonjiswe ukusebenza;uma kukhona noma iyiphi insalela ye-ion kasawoti, shiya ikholomu yokuhlanza ku-Buffer RW2 imizuzu engu-5 ekamelweni lokushisa futhi wenze i-centrifugation ukwandisa ukukhishwa kokungcoliswa kukasawoti.
2. Izinsalela ze-Ethanol ku-RNA elutioned.
Isincomo: Qinisekisa ukuthi ngemva kokugeza kwe-buffer RW2, yenza i-tube centrifugation operation engenalutho ngesivinini se-centrifugation esiboniswe ukusebenza, ukwandisa isikhathi sokusebenza kwe-tube centrifugation engenalutho kumaminithi angu-2 uma kusekhona insalela ye-ethanol, noma uyishiye ekamelweni lokushisa imizuzu engu-5 ngemva kwe-tube centrifugation engenalutho ukuze ukhulise ukukhishwa kwe-thanol.
