ISilwanyana sisonke se-RNA Isolation Kit Iyonke yeRNA yokutsalwa kunye neeKits zokuCoca kwiiTissues zezilwanyana kunye neSeli
Inkcazelo yeKit:
Ngokukhawuleza nangempumelelo ukukhupha ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwizicubu ezahlukeneyo zezilwanyana.
Akukho mfuneko yakukhathazeka malunga nokonakaliswa kwe-RNA.Yonke inkqubo i-RNase-Free
Susa ngokufanelekileyo i-DNA usebenzisa i-DNA-Cleaning Column
Susa i-DNA ngaphandle kokongeza i-DNase
Elula-yonke imisebenzi igqitywe kwiqondo lokushisa
Ukukhawuleza-ukusebenza kunokugqitywa kwimizuzu engama-30
Ikhuselekile-akukho reagent ephilayo esetyenzisiweyo
Ukucoceka okuphezulu-OD260 / 280≈1.8-2.1
Iinkcazelo Kits
50 Preps, 200 Preps
Le kit isebenzisa ispin ikholamu kunye nefomulaiphuhliswe yinkampani yethu, enokuthi ikhuphe ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwiintlobo zezilwanyana zezilwanyana kunye nokusebenza okuphezulu.Inika iKholamu ye-DNA-Cleaning esebenzayo, ekwazi ukwahlula ngokulula kwaye ibhengeze i-DNA ye-genomic kwi-supernatant kunye ne-tissue lysate, elula kunye neyonga ixesha;IKholamu ye-RNA kuphela inokubophelela ngokufanelekileyo i-RNA kwaye inokucutshungulwa ngaxeshanye ngefomula eyodwa Iisampuli ezininzi.
Yonke inkqubo i-RNase-Free, ukuze i-RNA ekhutshiweyo ingathotywanga;I-Buffer RW1, i-Buffer RW2 inkqubo yokuhlamba i-buffer, ukwenzela ukuba i-RNA efunyenweyo ayinayo iprotheni, i-DNA, i-ion, kunye nongcoliseko lwe-organic compound.
Amacandelo ekhithi
| Animal Total RNA Isolation Kit | ||
| Amacandelo ekhithi | RE-03011 | RE-03014 |
| 50 T | 200 T | |
| Isithinteli RL1* | 25ml | 100ml |
| Isithinteli RL2 | 15ml | 60ml |
| Isithinteli RW1* | 25ml | 100ml |
| Isithinteli RW2 | 24ml | 96ml |
| RNase-Free ddH2O | 10ml | 40ml |
| IKholam ye-RNA kuphela | 50 | 200 |
| Ikholamu yokucoca iDNA | 50 | 200 |
| Incwadi Yemiyalelo | Iqhekeza eli-1 | Iqhekeza eli-1 |
Ulwazi lwemveliso
| Ifomathi | Spin ikholamu | Icandelo lokucoca | Ikholamu ye-Foregene, i-reagent |
| Flux | 1-24 iisampulu | Ixesha lokulungiselela | ~30 min (iisampulu ezingama-24) |
| Centrifuge | Desk centrifuge | Ukuhlukana kwepyrolysis | Ukwahlukana kwe-Centrifugal |
| Isampulu | Izicubu zezilwanyana;iseli | Isixa seesampuli | Izicubu: 10-20 mg;Iseli:(1-5)×106 |
| Umthamo weElution | 50-200 μL | Ubuninzi bomthamo wokulayisha | 850 μL |
Iimpawu&izinto eziluncedo
■ Akukho mfuneko yakuzikhathaza ngokuthotywa kweRNA;yonke inkqubo i-RNase-Free
■ Ukususa ngokufanelekileyo i-DNA-usebenzisa i-DNA-Cleaning Column
■ Susa i-DNA ngaphandle kokufaka i-DNase
■ Ilula-yonke imisebenzi igqityezelwa kubushushu begumbi
■ Ukukhawuleza -ukusebenza kunokugqitywa kwimizuzu engama-30
∎ Ekhuselekileyo-akukho zixhobo zokuvuselela izinto eziphilayo ezifunekayo
■ Ukucoceka okuphezulu -OD260/280≈1.8-2.1
Isicelo seKit
Ilungele ukutsalwa kunye nokucocwa kwe-RNA iyonke ukusuka kwiintlobo ezahlukeneyo zezicubu zezilwanyana ezitsha okanye ezikhenkcezisiweyo okanye iiseli ezikhulisiwe.
Iiparamitha zemveliso
■ Izicelo ezisezantsi: i-first-strand cDNA synthesis, RT-PCR, i-molecular cloning, i-Northern Blot, njl.
■ Iisampulu: izihlunu zezilwanyana, iiseli ezikhuliswayo
■ Umthamo: Izicubu 10-20mg, Iiseli(2-5)×106
■ Ubuninzi be-DNA yokubopha umthamo wekholomu yokucoca: 80 μg
■ Umthamo we-Elution: 50-200 μl
Umzobo
Animal Total RNA Isolation Kit baphathwa 20mg
Iisampulu ezintsha zemouse, thatha i-5% esulungekileyo iyonke i-RNA 1% ye-agar
Glycogel electrophoresis
1: Udakada 2: Izintso
3: Isibindi 4: Intliziyo
Ukugcinwa kunye nobomi beshelufu
Ikhithi ingagcinwa iinyanga ezingama-24 kwiqondo lobushushu begumbi (15–25 ℃) okanye 2–8 ℃ ixesha elide.I-Buffer RL1 inokugcinwa kwi-4 ℃ kwinyanga eyi-1 emva kokongeza i-β-mercaptoethanol (ukhetho).
Amanqaku acatshulweyo
1.UKUBA:18.808:Zheng, Q., Qin, F., Luo, R., et al.I-mRNA-Elayishiweyo iLipid-NjengeNanoparticles yoHlelo oluSiseko seLiver ngokuPhuculwa koYilo oluDityanisiweyo oluPhakathi.Adv.Umsebenzi.Mater.2021, 31, 2011068.doi:10.1002/adfm.202011068.
2.UKUBA:18.187:Yena X, Hong W, Yang J, et al.I-apoptosis ezenzekelayo yeeseli ekulungiseleleni i-stem cell zonyango zenza i-immunomodulatory iziphumo ngokukhululwa kwe-phosphatidylserine.Ukuhanjiswa kweSignali ekuJoliswe kuyo.2021 Jul 14;6(1):270.doi: 10.1038/s41392-021-00688-z.
3.UKUBA: 17.97:Dai Z, Liu H, Liao J, et al.Ukuguqulwa kwe-N7-Methylguanosine tRNA kwandisa uguqulelo lwe-oncogenic mRNA kwaye ikhuthaza ukuqhubeka kwe-intrahepatic cholangiocarcinoma.Iseli yeMol.2021 Jul 29:S1097-2765 (21) 00555-4.doi: 10.1016/j.molcel.2021.07.003.
4.UKUBA:9.225:Cao X, Shu Y, Chen Y, et al.Ukuguqulwa kwe-M6A ye-Mettl14-Mediated Iququzelela ukuHlaziywa kwesibindi ngokuGcina i-Endoplasmic Reticulum Homeostasis.Iseli Mol Gastroenterol Hepatol.2021;12(2):633-651.doi: 10.1016/j.jcmgh.2021.04.001.
RNA isoaltion kits for eminye imithombo yesampuluziyafumaneka:
Iseli, isityalo, intsholongwane, igazi, njl.
I-RNA ayikhutshwa okanye izivuno ze-RNA ziphantsi
Kuhlala kukho izinto ezahlukeneyo ezichaphazela ukusebenza kakuhle kokubuyisela, njengale: umxholo we-RNA we-tissue, indlela yokusebenza, umthamo we-lution, njl.
1. Ibhafu ye-ice okanye i-cryogenic (4 ° C) i-centrifugation yenziwa ngexesha lokusebenza.
Isincomo: Sebenza kwiqondo lokushisa (15-25 ° C) kuyo yonke inkqubo, musa ukuhlamba i-ice kunye ne-centrifuge kumaqondo aphantsi.
2. Ukugcinwa kwesampulu engafanelekanga okanye ixesha lokugcina isampuli.
Isindululo: Gcina iisampulu kwi -80 °C okanye umkhenkce kwinitrogen engamanzi kwaye uthintele ukusetyenziswa okuphindaphindiweyo komkhenkce;zama ukusebenzisa izicubu ezitsha okanye iiseli ezikhuliswayo zokutsalwa kwe-RNA.
3. I-lysis yesampuli eyaneleyo.
Isincomo: Xa i-homogenizing tissue, qinisekisa ukuba i-tissue i-homogenized ngokwaneleyo kwaye iiseli zethishu zahlulwe ngokwaneleyo ukucacisa ukukhululwa kwe-RNA.
4. I-eluent ayidityaniswa ngokuchanekileyo.
Ingcebiso: Qinisekisa ukuba i-RNase-Free ddH2I-O yongezwa kwi-dropwise phakathi kwe-membrane yekholomu yokucoca.
5. Umthamo ochanekileyo we-absolute ethanol awuzange wongezwe kwi-Buffer RL2 okanye kwi-Buffer RW2.
Ingcebiso: Landela imiyalelo, yongeza umthamo ochanekileyo we-ethanol epheleleyo kwi-Buffer RL2 kunye ne-Buffer RW2 kwaye udibanise kakuhle phambi kokusebenzisa ikhithi.
6. Idosi yesampula yezicubu ayifanelekanga.
Isincomo: Sebenzisa i-10-20 mg yezicubu okanye (1-5) × 106iiseli nge-500 μl buffer RL1, njengoko ukusetyenziswa kwethishu ngokugqithisileyo kunokubangela ukutsalwa kwe-RNA okucuthiweyo.
7. Umthamo ongafanelekanga okanye i-elution engaphelelanga.
Isincomo: Umthamo we-elution wekholamu yokucoca yi-50-200 μl;ukuba isiphumo se-elution asanelisi, kuyacetyiswa ukuba kwandiswe ixesha lokubeka ubushushu begumbi emva kokongeza i-RNase-Free ddH efudunyeziweyo.2O, umz. kwi-5-10 min.
8.Ikholamu yokucoca inentsalela ye-ethanol emva kokuhlamba kwe-Buffer RW2.
Isincomo: Ukuba kukho intsalela ye-ethanol emva kokuhlamba kwe-Buffer RW2, i-tube engenanto ye-centrifugation ye-1min, ixesha le-tube engenanto yokusebenza kwe-centrifugation linganyuswa ukuya kwi-2min, okanye ikholomu yokucoca inokubekwa kwindawo yokushisa ye-5 min ukususa ngokwaneleyo i-ethanol eseleyo.
I-RNA ecocekileyo ithotywa
Umgangatho we-RNA ecocekileyo inxulumene nezinto ezifana nokugcinwa kwesampulu, ukungcoliswa kwe-RNase, kunye nokukhwabanisa, njl.
1. Iisampulu zezicubu azigcinwanga ngexesha.
Isincomo: Ukuba iisampulu zethishu okanye iiseli azisetyenziswanga ngexesha elifanelekileyo emva kokuqokelelwa, ngoko nangoko gcina i-cryopreserve kwi -80 °C okanye initrogen engamanzi.Ukukhupha i-RNA, sebenzisa ithishu esanda kuthathwa okanye isampulu yeseli xa kunokwenzeka.
2. Ukunyibilika okuphindaphindiweyo kweesampuli zethishu.
Isincomo: Xa ugcina iisampulu zethishu, kungcono ukuba uzinqumle zibe ngamaqhekeza amancinci ukuze zigcinwe, kwaye ususe esinye seziqwenga xa uzisebenzisa ukuphepha ukunyibilika okuphindaphindiweyo kwesampulu kunye nokuthotywa kwe-RNA.
3. I-RNase yaziswa okanye ayinxibi iiglavu ezilahlwayo, iimaski, njl njl ngexesha lokusebenza.
Isincomo: Imifuniselo yokutsalwa kwe-RNA yenziwa ngcono kumagumbi okukhohlisa e-RNA kwaye itafile iyacinywa phambi kovavanyo.
Nxiba iiglavu ezilahlwayo kunye nemaski ngexesha lovavanyo lokunciphisa ukuthotywa kwe-RNA okubangelwa kukwaziswa kwe-RNase.
4. Ii-reagents zingcolisekile nge-RNase ngexesha lokusetyenziswa.
Ingcebiso: Faka endaweni entsha yeSilwanyana esipheleleyo se-RNA ye-Isolation Kit ukulungiselela imifuniselo ehambelanayo.
5. Iityhubhu ze-centrifuge, iingcebiso, njl.njl ezisetyenziswa kwi-RNA manipulation zingcolile nge-RNase.
Ingcebiso: Qinisekisa ukuba iityhubhu ze-centrifuge, iingcebiso, iipipette, njl.njl ezisetyenziswa kwi-RNA extraction azinayo yonke i-RNase.
I-RNA esulungekileyo ichaphazela imifuniselo esezantsi
I-RNA ihlanjululwe yikholomu yokucoca, ukuba iiyoni zetyuwa, umxholo weprotheyini mkhulu kakhulu uya kuchaphazela umfuniselo osezantsi, onje: uguqulelo lombhalo, Northern Blot et al.
1. I-RNA ekhutshiweyo ineentsalela ze-ion yetyuwa.
Isincomo: Qinisekisa ukuba umthamo ochanekileyo we-ethanol wongezwe kwi-Buffer RW2 kwaye wenze i-2 ikholamu yokuhlanjululwa kwi-speed ye-centrifugal eboniswe ekusebenzeni;ukuba kukho nayiphi na intsalela ye-ion yetyuwa, shiya ikholomu yokucoca kwi-Buffer RW2 ye-5 min kwiqondo lokushisa elingaphakathi kwaye wenze i-centrifugation ukwandisa ukukhutshwa kwetyuwa.
2. Intsalela ye-Ethanol kwi-RNA elutioned.
Isincomo: Qinisekisa ukuba emva kokuhlamba i-buffer RW2, yenza i-tube engenanto ye-centrifugation operation kwisantya se-centrifugation esiboniswe ukusebenza, ukwandisa ixesha le-tube engenanto ye-centrifugation operation ukuya kwi-2 min ukuba kusekho i-ethanol intsalela, okanye uyishiye kwindawo yokushisa kwe-5 min emva kwe-tube engenanto ye-centrifugation yokwandisa ukususwa kwe-ethanol ye-ethanol.
