RNase nijambo ryumvikana abanyeshuri benshi bakunze gukora ubushakashatsi bwo gukuramo RNA badashaka kumva.Intwaro zuzuye, RNA yaje gukururwa hamwe na reagent zifite ubumara bukabije fenol na chloroform yarangiritse.Ntabwo niyunze!!!Uyu munsi, reka turebe inkomoko ya Rnase izwi.

Ribonuclease (RNase), cyangwa RNase, ni nuclease ishobora hydrolyze RNA muri molekile nto.RNase, nka poroteyine ntoya, ihagaze neza bidasanzwe .Ubushyuhe bwo hejuru busanzwe hamwe numuvuduko ukabije wamazi sterilisation hamwe na protein inhibitor ntishobora kubikora rwose.Guhagarara kwa RNase ahanini biva kumurongo wa disulfide muburyo.Kurugero, RNase ikunze gukoreshwa na bovine pancreas ifite aside amine 124 gusa, ariko irimo 4 disulfide.Inkunga ya sulfure hamwe na disulfide ihuza RNase hamwe nubushyuhe buhebuje.Mubyongeyeho, rnase ifite uburemere buke bwa molekuline kandi irashobora kugarura vuba ihinduka ryayo ryambere mubihe byinshi.

Usibye kuba uhagaze neza cyane,RNase iragaragara hose muri laboratoire .RNase nuburyo bwo kwirinda ibinyabuzima.Kuri selile, RNA idasanzwe ikica.Ugereranije na ADN idasanzwe, RNA idasanzwe akenshi iba iteje akaga.RNA yandukuwe kandi irahindurwa, kuburyo ibinyabuzima hafi ya byose byahinduye RNase kugirango birinde igitero cya RNA idasanzwe.Noneho rero, selile ziterwa na laboratoire nawe ukuramo RNA usohora impumuro ya RNase.Amazi yumubiri wumuntu (amacandwe, amarira, nibindi) arimo RNase nyinshi, ntukarire rero mugihe RNA yangiritse.Uko urira, niko kwangirika kwa RNA ari bibi!!Mushikiwabo Daiyu ntabwo akwiriye gukuramo RNA!

Byongeye kandi, uruhu rwawe rworoshye kandi rurimo RNase nyinshi, kandi marikeri, pipeti, inzugi za firigo, hamwe ninzugi zumuryango zakozweho nuruhu zirimo na RNase.

Hamwe no gutombora cyane, reka turebe uko twakemura RNase.

Ikintu cya mbere abantu bose batekereza mugihe cyo gukuraho RNA niDEPC(diethyl pyrocarbonate).DEPC yerekana cyane poroteyine ihuza impeta ya imidazole ya RNase ikora itsinda rya histidine, bityo bikabuza ibikorwa bya enzyme.0.1% DEPC irashobora kugira ingaruka nziza zo gukuraho kuri Rnase, ariko dukeneye kwitondera ko DEPC ari kanseri izwi, bityo rero tugomba kwitondera bidasanzwe mugihe uyikoresheje.

Kuri RNase, dukeneye guhera kubintu bibiri,icyambere nukubuza ibikorwa bya endogenous RNase

Imyanda isanzwe ya RNA ikuramo nka guanidine isothiocyanate na DTT ikubiye muri Trizol irashobora gufungura umurongo wa disulfide wa RNase, ariko haracyari RNase zimwe na zimwe, cyane cyane mubyitegererezo, bityo rero witondere ubushyuhe buke.

1.Ako kanya winjize icyitegererezo cya tissue muri azote yuzuye nyuma yo kuyikuramo, cyangwa mubisubizo byubucuruzi bwa RNA.

2.Nyuma yo gukuramo RNA yicyitegererezo cyakagari, ongeraho igisubizo cya lysis hanyuma uyitere kumasanduku ya barafu

3. Nibyiza gukoresha azote yuzuye yo gusya mugihe ingero za tissue zihuriweho.Mugihe ukoresheje amashanyarazi ya homogenizer idafite azote yuzuye, witondere mbere yo gukonjesha byimazeyo adaptate ya homogenate.

Iya kabiri ni DNase idasanzwe

1.Ba intwaro zuzuye, wambare ikote rya laboratoire, wambare mask, kandi urebe neza ko wambara uturindantoki dushya (ntukoreshe neza !! Twabibutsa ko Trizol ishobora kwangirika cyane, kandi nayo irakomeye cyane binyuze mu ntoki, ntugatonywe ku biganza).

2.Inama zose zikoreshwa, imiyoboro ya EP, imiyoboro ya PCR nibindi bikoresho bigomba kuvurwa de-RNase.Irashobora gushirwa muri 0.1% DEPC hanyuma igashyirwaho igitutu munsi yumuvuduko mwinshi.Witondere imikorere muri fume hood.Abanyagitugu baho barashobora kugura byimazeyo ibikoreshwa mugukuraho enzymes.

PS: Reka nkubwire uburyo bwubunebwe.Nubwo ubushyuhe bwinshi numuvuduko mwinshi bidashobora gukuraho RNase rwose, bizakuraho igice kinini cyacyo.Inshuro 2 z'ubushyuhe bwinshi n'umuvuduko mwinshi bigira ingaruka nziza, kandi gukuramo RNA ntacyo bigira.

3.Inzoga zirashobora gutandukanya poroteyine,ameza yo gukuramo RNA rero ashobora guhanagurwa na 75% alcool , na gants nazo zirashobora guterwa inzoga.

4.Umuti wa nyuma wa RNA ushonga hamwe na centrifuge tube nayo igomba kuvurwa de-RNase.Amazi ya DEPC ni igisubizo gikunze gukoreshwa RNA ishonga.Reka tuvuge uburyo bwiza bwo gutegura amazi ya DEPC (ibuka gusubiramo ibicuruzwa DEPC yubucuruzi mugihe uyigura)

Ongeramo DEPC mumazi ya ultrapure kuri 1: 1000, uzunguze neza, ureke uhagarare ijoro kuri 37 ° C, hanyuma uhagarike kuri 121 ° C munsi yubushyuhe bwinshi numuvuduko mwinshi muminota 15.Amazi ya DEPC arashobora kubikwa kuri -20 ° C muri 1ml aliquots.

Hanyuma, muri make: ubushyuhe buke nurufunguzo, ibikoreshwa bifatwa neza, bitwaje intwaro byuzuye kandi bike!

Nibyiza, nibyo kubikorwa byuyu munsi.Nkwifurije kwibanda kuri RNA byose hamwe nubuziranenge wavuze.Byombi A260 / A280 ni 2.0!!!

Birumvikana, niba ukoresha aicyumba cy'ubushyuhe bwo gukora ibikoresho byo gukuramo RNA, ntushobora guhura nibibazo byavuzwe haruguru.

Gukorera mubushyuhe bwicyumba, utongeyeho DNase, ikuramo RNA yose muri selile muminota 11, ikanakuramo RNA yose mubice byinyamanswa cyangwa ibimera muminota 30.

Kuburugero rwikigereranyo, nyamuneka hamagara:overseas@foregene.com

Ibicuruzwa bifitanye isano:

https://www.foreivd.com/cell-total-rna-isolation-kit-product/

https://www.foreivd.com/ibisanzwe-total-rna-isolation-kit-product/

https://www.foreivd.com/plant-total-rna/