Xirmooyinka Go'doominta RNA Wadarta Xayawaanada
Sharaxaada Qalabka:
Si degdeg ah oo hufan uga soo saar wadarta guud ee RNA nadiif-sare iyo tayo sare leh oo laga soo saaray unugyo xayawaanno kala duwan.
Looma baahna in laga walwalo xaalufka RNA.Nidaamka oo dhan waa RNase-free
Si wax ku ool ah uga saar DNA adoo isticmaalaya Tiirka Nadiifinta DNA
Ka saar DNA adigoon ku darin DNA-ga
Fudud - dhammaan hawlgallada waxaa lagu dhammeeyaa heerkulka qolka
Dhaqso-qalliinka waxa lagu dhamayn karaa 30 daqiiqo gudahood
Badbaadsan — lama isticmaalin reageent organic
Nadaafadda sare-OD260/280≈1.8-2.1
Qeexitaannada Qalabka
50 Diyaarin, 200 Diyaarin
Qalabkani waxa uu isticmaalaalafdhabarta iyo caanahaoo ay soo saartay shirkadeena, taas oo ka soo saari karta wadarta guud ee nadiifka ah iyo tayada sare ee RNA laga soo bilaabo unugyo xayawaan ah oo kala duwan oo leh waxtar sare leh.Waxay bixisaa DNA-Nadiifinta Tiirka wax ku ool ah, kaas oo si sahlan u kala saari kara oo u xajin kara DNA-da genomic-ka sare iyo unugyada lysate, fudud iyo waqti-badbaadinta;Tiirka RNA-keliya ayaa si hufan u xidhi kara RNA waxaana lagu farsamayn karaa qaacido gaar ah Tiro badan oo muunado ah.
Nidaamka oo dhan waa RNase-free, si RNA-ga la soo saaray aanu hoos u dhicin;Buffer RW1, Buffer RW2 nidaamka dhaqida kaydinta, si RNA-ga la helay uu xor uga ahaado borotiinka, DNA, ion, iyo wasakheynta isku dhafka dabiiciga ah.
Qaybaha kitka
| Wadarta Xayawaanka Qalabka Go'doonka RNA | ||
| Qaybaha kitka | RE-03011 | RE-03014 |
| 50 T | 200 T | |
| kaydka RL1* | 25ml | 100ml |
| Buffer RL2 | 15ml | 60ml |
| Bakhaar RW1* | 25ml | 100ml |
| Buffer RW2 | 24ml | 96ml |
| RNase-free ddH2O | 10ml | 40ml |
| Tiirka RNA-kaliya | 50 | 200 |
| Tiirka-Nadiifinta DNA | 50 | 200 |
| Buugga tilmaamaha | 1 gabal | 1 gabal |
macluumaadka alaabta
| Qaabka | Tiirka lafdhabarta | Qaybta nadiifinta | Tiirka Foregene, reagent |
| Flux | 1-24 muunado | Waqtiga diyaargarowgaba | ~ 30 daqiiqo (24 muunado) |
| Centrifuge | Xarunta dhexe ee miiska | Kala saarista pyrolysis | Kala soocida centrifugal |
| Tusaale | Nudaha xoolaha;unugga | Tirada muunada | Nudaha: 10-20 mg;Unug: (1-5)×106 |
| Mugga ilbaxnimada | 50-200 μL | Mugga rarida ugu badan | 850 μL |
Tilmaamaha & faa'iidooyinka
■ Looma baahna in laga walwalo xaalufka RNA;nidaamka oo dhan waa RNase-free
■ Si wax ku ool ah uga saar DNA-da adoo isticmaalaya Tiirka Nadiifinta DNA
■ Ka saar DNA adigoon ku darin DNA-da
■ Hawlgallada fudud-dhammaan waxaa lagu dhammeeyaa heerkulka qolka
■ Hawlgalka degdegga ah waxa lagu dhamayn karaa 30 daqiiqo
■ Badbaado-ma baahna reageent organic ah
■ Nadaafadda sare -OD260/280≈1.8-2.1
Codsiga qalabka
Waxay ku habboon tahay soo saarista iyo nadiifinta wadarta RNA ee noocyo kala duwan oo unugyo xayawaan ah oo cusub ama barafaysan ama unugyo dhaqan leh.
Xuduudaha alaabta
■ Codsiyada hoose: isku dhafka cDNA ee ugu horreeya, RT-PCR, cloning molecular, Northern Blot, iwm.
Tusaalooyinka: unugyada xoolaha, unugyo dhaqan
■ Qiyaasta: Unugyada 10-20mg, Unugyada (2-5)×106
■ Awoodda ugu badan ee DNA-da ee tiirka sifaynta: 80 μg
■ Mugga iftiinka: 50-200 μl
Jaantuska
Wadarta Xayawaanka Go'doominta RNA ee lagu daweeyay 20mg
Muuska cusub, qaado 5% wadarta guud ee RNA la safeeyey 1% agar
Glycogel electrophoresis
1: beeryarada 2: kelyaha
3: Beerka 4: Wadnaha
Kaydinta iyo nolosha shelf
Qalabka waxaa lagu kaydin karaa 24 bilood heerkulka qolka (15-25 ℃) ama 2-8 ℃ wakhti dheer.Buffer RL1 waxa lagu kaydin karaa 4 ℃ 1 bil kadib marka lagu daro β-mercaptoethanol (ikhtiyaar).
Maqaallo la soo xigtay
1.HADDII:18.808:Zheng, Q., Qin, F., Luo, R., iyo al.mRNA-Loaded Lipid-Lipid Nanoparticles oo loogu talagalay Tafatirka Saldhigga Beerka iyadoo loo marayo Hagaajinta Naqshadaynta Isku-dhafka Dhexe.Adv.ShaqaaleMater.2021, 31, 2011068.doi:10.1002/adfm.202011068.
2.HADDII:18.187:He X, Hong W, Yang J, iyo al.apoptosis kediska ah ee unugyada ku jira diyaarinta unugyada asliga ah ee daweynta waxay sameeyaan saameyn immunomodulatory ah iyada oo la sii daayo fosfatidylserine.Bartilmaameedka Is-beddelka Signal Ther.2021 Jul 14;6(1):270.doi: 10.1038/s41392-021-00688-z.
3.HADDII:17.97:Dai Z, Liu H, Liao J, iyo al.N7-Methylguanosine tRNA wax ka beddelka waxay wanaajisaa tarjumaada mRNA oncogenic waxayna kor u qaadaa horumarka cholangiocarcinoma intrahepatic.Mol Cell.2021 Jul 29:S1097-2765(21)00555-4.doi: 10.1016/j.molcel.2021.07.003.
4.HADDII:9.225:Cao X, Shu Y, Chen Y, iyo al.Mettl14-Mediated m6A Beddelka ayaa Fududeeya Dib u soo Kabashada Beerka iyadoo la ilaalinayo Endoplasmic Reticulum Homeostasis.Unugyada Mol Gastroenterol Hepatol.2021;12 (2): 633-651.doi: 10.1016/j.jcmgh.2021.04.001.
Xirmooyinka go'doominta RNA ee ilo muunado kalewaa la heli karaa:
Unug, dhir, fayras, dhiig, iwm.
RNA lama soo saarin ama wax soo saarka RNA waa yar yahay
Inta badan waxaa jira arrimo kala duwan oo saameeya waxtarka soo kabashada, sida: muunada unugyada RNA content, habka hawlgalka, mugga elution, iwm.
1. Barafka qubeyska ama cryogenic (4°C) centrifugation ayaa la sameeyay intii lagu jiray qaliinka.
Talo soo jeedin: Ku shaqee heerkulka qolka (15-25 ° C) inta lagu jiro habka oo dhan, ha ku maydhin baraf oo ha ku daboolin heerkul hoose.
2. Ilaalinta muunad khaldan ama wakhtiga kaydinta muunada xad dhaafka ah.
Talo-soo-jeedin: Ku kaydi shay-baarka--80 °C ama ku qabooji nitrogen dareeraha ah kana fogow isticmaalka baraf-dhalashada ee soo noqnoqda;isku day inaad u isticmaasho unug cusub ama unugyo dhaqan leh soo saarista RNA.
3. Shaybaar aan ku filnayn.
Talo soo jeedin: Marka unugyadu isku mid yihiin, hubi in unuggu si ku filan isu ekaysiiyo iyo in unugyadu si ku filan u kala qaybsamaan si loo sharaxo sii daynta RNA.
4. Eluent si sax ah looguma darin.
Talo soo jeedin: Xaqiiji in RNase-free ddH2O waxaa lagu daraa hoos udhaca dhexe ee xuubka tiirka sifaynta.
5. Mugga saxda ah ee ethanol buuxa laguma darin Buffer RL2 ama Buffer RW2.
Talo soo jeedin: Raac tilmaamaha, ku dar mugga saxda ah ee ethanol buuxa ee Buffer RL2 iyo Buffer RW2 oo si fiican isku qas ka hor intaadan isticmaalin qalabka.
6. Qiyaasta muunadda unugyada ma habboona.
Talo soo jeedin: Isticmaal 10-20 mg oo unug ah ama (1-5) × 106unug kasta 500 μl baffer RL1, maadaama isticmaalka nudaha xad-dhaafka ah ay keeni karto soo saarista RNA oo yaraada.
7. Mugga sare-u-qaadista oo aan sax ahayn ama muuqaal aan dhammaystirnayn.
Talo soo jeedin: Mugga sare ee tiirka nadiifinta waa 50-200 μl;Haddii saamaynta sare-u-qaadista aysan ku qanacsanayn, waxaa lagu talinayaa in la kordhiyo waqtiga meelaynta heerkulka qolka ka dib marka lagu daro RNase-free ddH preheated2O, tusaale ahaan 5-10 daqiiqo
8.Tiirka nadiifinta ayaa leh hadhaaga ethanol ka dib markii la dhaqo Buffer RW2.
Talo soo jeedin: Haddii ay jirto hadhaaga ethanol ka dib markii la dhaqo Buffer RW2, centrifugation tube madhan 1min, wakhtiga hawlgalka centrifugation tuubada madhan waxaa la kordhin karaa ilaa 2min, ama tiirka sifaynta waxaa la dhigi karaa heerkulka qolka 5 min si ku filan looga saaro ethanol hadhaaga ah.
RNA la safeeyey ayaa hoos u dhacay
Tayada RNA la safeeyey waxay la xiriirtaa arrimo ay ka mid yihiin ilaalinta shaybaarka, faddaraynta RNase, iyo khalkhalgelinta, iwm.
1. Tijaabada unugyada lama hayo wakhtigeeda.
Talo soo jeedin: Haddii shaybaarada unugyada ama unugyada aan loo isticmaalin waqti ku habboon ka dib ururinta, isla markiiba cryopreserve at -80 °C ama nitrogen dareere ah.Si aad u soo saarto RNA, isticmaal unug cusub oo la qaaday ama muunad unug mar kasta oo ay suurtogal tahay.
2. Baraf-dhalaalitaan soo noqnoqda ee shaybaarada unugyada.
Talo soo jeedin: Marka la kaydinayo shaybaarka unugyada, waxa fiican in la jarjaro qaybo yaryar si loo xafido, oo laga saaro mid ka mid ah qaybaha marka la isticmaalayo si looga fogaado baraf-dhalashada soo noqnoqda ee muunada iyo hoos u dhaca RNA.
3. RNase waa la soo bandhigay ama aan la xidhin galoofisyada la tuuri karo, waji-xidhka, iwm. inta lagu jiro qaliinka.
Talo soo jeedin: Tijaabooyinka soo saarista RNA waxaa sida ugu fiican loogu sameeyaa qolal kala duwan oo wax lagu sameeyo RNA miiskana waa la nadiifiyaa ka hor tijaabada.
Xiro galoofyada iyo maaskaro la tuuri karo inta lagu jiro tijaabada si loo yareeyo xaalufka RNA ee ay keento hordhaca RNase.
4. Reagen-yada waxa ku wasakhaysan RNase marka la isticmaalayo.
Talo-soo-jeedin: Ku beddel Xayawaanada cusub ee Wadarta Guud ee Go'doonka RNA ee tijaabooyinka la xidhiidha.
5. Tuubooyinka centrifuge, talooyinka, iwm. ee loo isticmaalo wax-ka-beddelka RNA waxay ku wasakhoobeen RNase.
Talo soo jeedin: Xaqiiji in tuubbooyinka centrifuge, talooyinka, pipettes, iwm. ee loo isticmaalo soo saarista RNA ay dhamaantood yihiin kuwo ka madax banaan RNase.
RNA la safeeyey ee la helay waxay saamaysaa tijaabooyinka hoose
RNA oo lagu nadiifiyo tiirka sifaynta, haddii ions milix ah, waxa ku jira borotiinku aad u weyn yahay waxay saamaynaysaa tijaabada hoose, sida: qoraalka dib u noqoshada, Northern Blot et al.
1. RNA-ga la macmalay waxa uu leeyahay hadhaaga ion cusbo.
Talo soo jeedin: Xaqiiji in mugga saxda ah ee ethanol lagu daray Buffer RW2 oo samee 2 tiir nadiifin ah oo dhaqida xawliga centrifugal ee lagu tilmaamay hawlgalka;Haddii ay jiraan wax hadhaaga ion cusbo, u dhaaf tiirka sifaynta Buffer RW2 5 daqiiqo heerkulka qolka oo samee centrifugation si aad u kordhiso ka saarida wasakhowga milixda.
2. Hadhaaga Ethanol ee RNA-ga la macmalay.
Talo soo jeedin: Xaqiiji in ka dib marka la dhaqo RW2 baffer, samee hawlgalka centrifugation tube madhan xawaaraha centrifugation ee lagu tilmaamay hawlgalka, kordhiyo waqtiga hawlgalka centrifugation tube madhan 2 min haddii ay weli jiraan haraaga ethanol, ama ka tago heerkulka qolka 5 daqiiqo ka dib tuubada centrifugation madhan si loo kordhiyo ka saarida ethanolue.
