I-Sterrilization yeengcebiso ze-pipette kunye neetyhubhu ze-EP, njl.

1. Lungisa i-0.1% (iwaka) i-DEPC (into enetyhefu ephezulu) ngamanzi adibeneyo, yisebenzise ngononophelo kwi-fume hood, kwaye uyigcine kwi-4 ° C kude nokukhanya;

Amanzi e-DEPC ngamanzi acocekileyo aphathwa nge-DEPC kwaye acociweyo bubushushu obuphezulu kunye noxinzelelo oluphezulu.Ivavanywe ukuba ayinayo i-RNase, i-DNase kunye ne-proteinase.

2. Beka i-pipette tip kunye ne-EP tube kwi-0.1% DEPC, kwaye uqinisekise ukuba i-pipette tip kunye ne-EP tube izaliswe nge-0.1% DEP.

3. Khusela ekukhanyeni, yima, ubusuku bonke (12-24h)

4. Ibhokisi equkethe i-tip kunye ne-EP tube ayifuni ukuba ifakwe kwi-DEPC.Emva kokususa ngokungqongqo amanzi e-DEPC kwincam okanye ityhubhu ye-EP, yipakishe kwaye uyisonge.

5. 121 degrees Celsius, 30min

6. 180 degrees Celsius, yome iiyure ezininzi (ubuncinci iiyure ezi-3)

Qaphela: a.Nxiba iiglavu zelatex kunye nemaski xa uphethe iDEPC!b, okanye ngaphandle kwe-DEPC inzala, i-130 ℃, i-90min autoclave (iilabhoratri ezininzi zokuvala ubushushu obuphindwe kabini)

Iingqwalasela zokutsalwa kwe-RNA

Izinto ezimbini eziphambili zokungaphumeleli kwe-RNA yezicubu

Ukuthotywa kwe-RNA kunye neentsalela zokungcola kwizicubu,malunga nokuthotywa, masiqale sijonge ukuba kutheni i-RNA ekhutshwe kwiiseli ezikhuliswayo ingonakaliswa ngokulula.Izixhobo ezikhoyo zokukhutshwa kwe-RNA zonke ziqulethe amacandelo anqanda i-RNase ngokukhawuleza.Yongeza i-lysate kwiiseli ezikhuliswe, kwaye udibanise ngokulula, zonke iiseli zinokuxutywa ngokucokisekileyo kunye ne-lysate, kwaye iiseli zixutywe ngokupheleleyo.Emva kokuba iiseli zihlanjululwe, izithako ezisebenzayo kwi-lysate zithintela ngokukhawuleza i-intracellular RNase, ngoko ke i-RNA ihleli.Oko kukuthi, ngenxa yokuba iiseli ezikhuliswe ngokulula kwaye ziqhagamshelwe ngokupheleleyo kunye ne-lysate, i-RNA yazo ayithotywanga lula;kwelinye icala, i-RNA kwithishu ithotywa ngokulula ngenxa yokuba iiseli ezikwithishu akulula ukuba ziqhagamshelane ngokukhawuleza ne-lysate.ngenxa yoqhagamshelwano olwaneleyo.Ngoko,ucinga ukuba kukho indlela yokuguqula izicubu zibe yiseli enye ngelixa uvimbela umsebenzi we-RNA, ingxaki yokuthotywa ingasonjululwa ngokupheleleyo.

Ulwelo lokusila initrogen yeyona ndlela isebenzayo.Nangona kunjalo, indlela yolwelo yokusila initrogen inzima kakhulu, ngakumbi xa inani leesampuli likhulu.Oku kubangele eyona nto ilungileyo ilandelayo: i-homogenizer.Ii-homogenizerindlela ayicingi ngombuzo wokuba umsebenzi we-RNase uthintelwe njani ngaphambi kokuba iiseli zidityaniswe ne-lysate, kodwa kunoko uthandazela ukuba izinga lokuphazamiseka kwezicubu ngokukhawuleza kunomlinganiselo apho i-intracellular RNase ithoba iRN.

Impembelelo ye-homogenizer yombane ingcono,kunye nempembelelo ye-homogenizer yeglasi ihlwempuzekileyo, kodwa ngokubanzi, indlela ye-homogenizer ayikwazi ukuthintela i-degradation phenomenon.Ngoko ke, ukuba i-extraction iyancipha, i-homogenizer yombane yasekuqaleni kufuneka isetyenziswe ukugaya nge-nitrogen engamanzi;i-homogenizer yeglasi yoqobo kufuneka itshintshwe ibe yi-homogenizer yombane okanye icolwe ngokuthe ngqo ngenitrogen engamanzi.Ingxaki iphantse ibe nokwenzeka nge-100%.zisombulule.

Ingxaki yentsalela yokungcola echaphazela imifuniselo elandelayo inezizathu ezininzi ezahlukahlukeneyo kunokuthotywa, kwaye izisombululo ziyahluka ngokuhambelanayo.Ukuququmbela,ukuba kukho ukuthotywa okanye ukungcola okushiyekileyo kwi-tissue, indlela yokukhupha / i-reagent yezinto ezithile zokulinga kufuneka ziphuculwe.Akunyanzelekanga ukuba usebenzise iisampulu zakho ezixabisekileyo ukuze uphucule: unokuthenga izilwanyana ezincinci ezifana nentlanzi/inkukhu emarikeni, thatha indawo ehambelanayo yemathiriyeli yokutsalwa kwe-RNA, kunye nenye indawo yokutsalwa kweprotein – sila ngomlomo, isisu kunye namathumbu.

I-RNA ekujoliswe kuyo ye-RNA ekhutshiweyo isetyenziselwa iimvavanyo ezahlukeneyo zokulandelelana, kwaye iimfuno zayo zomgangatho zahlukile

Ukwakhiwa kwethala leencwadi le-cDNA kufuna ingqibelelo ye-RNA ngaphandle kweentsalela ze-enzyme reaction inhibitors;Umntla ufuna ingqibelelo ephezulu ye-RNA kunye neemfuno ezisezantsi zeentsalela ze-enzyme reaction inhibitors;I-RT-PCR ayifuni ingqibelelo ephezulu kakhulu ye-RNA,kodwa inqanda ukusabela kwe-enzyme.Iimfuno zentsalela zingqongqo.Igalelo limisela imveliso;ngalo lonke ixesha injongo kukufumana eyona RNA yococeko iphezulu, iya kuxabisa abantu kunye nemali.

Ukuqokelelwa/ukugcinwa kweisampulu

Izinto ezichaphazela ukuthotywa emva kokuba isampulu ishiya umzimba ophilayo / okanye indawo yokukhula yantlandlolo, i-endo native enzymes kwisampulu iya kuqala ukuthoba i-RNA,kwaye izinga lokuthotywa linxulumene nomxholo we-endogenous enzymes kunye nobushushu.Ngokwesiko, kukho iindlela ezimbini kuphela zokuthintela ngokupheleleyo umsebenzi we-endogenous enzyme: yongeza i-lysate ngokukhawuleza kwaye i-homogenize ngokucokisekileyo kwaye ngokukhawuleza;Sika sibe ngamaqhekeza amancinci kwaye ngoko nangoko umkhenkce kwinitrogen engamanzi.Zombini iindlela zifuna ukusebenza ngokukhawuleza.Le yokugqibela ifanelekile kuzo zonke iisampuli, ngelixa i-yangaphambili ifanelekile kuphela kwizicubu ezinomxholo ophantsi weeseli kunye ne-endogenous enzymes kwaye kulula ukwenza i-homogenize.Ngokukodwa, izicubu zesityalo, isibindi, i-thymus, i-pancreas, i-spleen, ingqondo, amafutha, izicubu zomzimba, njl.

Ukuqhekeka kunye ne-homogenization yeesampuli

Imiba echaphazela ukuthotywa kunye nokuqhekeka kweSampuli yeSivunokwi-homogenization ngokucokisekileyo, eyeyokukhutshwa okupheleleyo nokupheleleyo kwe-RNA.Iiseli zinokudityaniswa ngokuthe ngqo kwi-homogenized ngaphandle kokwaphulwa.Izicubu ziyakwazi ukuba zibe homogenized kuphela emva kokuba zaphukile.Igwele kunye neebhaktheriya kufuneka zaphulwe ngee-enzyme ezihambelanayo ngaphambi kokuba zibe ne-homogenized.Izicubu ezinomxholo ophantsi we-enzyme ye-endogenous kunye ne-homogenization elula inokutyunyuzwa kwaye i-homogenized ngexesha elinye kwi-lysate yi-homogenizer;Izicubu zesityalo, isibindi, i-thymus, i-pancreas, i-spleen, ingqondo, amafutha, izicubu zomzimba kunye nezinye iisampulu, ziphezulu kwi-endogenous enzymes okanye akulula ukuba zibe homogenized,ngoko ukuphazamiseka kwezicubu kunye ne-homogenization kufuneka kwenziwe ngokwahlukileyo.Eyona ndlela inokwethenjelwa kunye neyona ndlela ivelisa kakhulu yokuqhekeka kukugaya nge-nitrogen engamanzi, kwaye eyona ndlela ithembekileyo ye-homogenization kukusetyenziswa kwe-homogenizer yombane.Inqaku elikhethekileyo malunga nokusila ngenitrogen engamanzi: isampulu akufuneki inyibilikiswe ngexesha lonke lenkqubo yokusila, njengoko i-endogenous enzymes kunokwenzeka ukuba isebenze xa ingumkhenkce.

Ukhetho lwe-lysate

Ukuchaphazela ukusebenziseka lula kunye nezinto ezishiyekileyo zokungcola kwe-endogenous Izisombululo ezisetyenziswa ngokuqhelekileyo ze-lysis zinokuphantse zithintele umsebenzi we-RNase.Ngoko ke, inqaku eliphambili lokukhetha isisombululo se-lysis kukuqwalasela ngokudibanisa nendlela yokucoca.Kukho ngaphandle enye:iisampulu ezinomxholo ophezulu we-endogenous enzyme ziyacetyiswa ukuba zisebenzise i-lysate equlethe i-phenol ukwandisa amandla okungasebenzi kwe-endogenous enzymes.

Ukukhetha indlela yokucoca

Izinto ezichaphazela ukungcola okushiyekileyo kwe-endogenous, isantya sokutsalwa kwiisampuli ezicocekileyo ezifana neeseli, iziphumo ezanelisayo zinokufumaneka phantse nayiphi na indlela yokucoca ekhoyo.Kodwa kwezinye iisampulu ezininzi, ngakumbi ezo zinamanqanaba aphezulu okungcola okunje ngezityalo, isibindi, ibhaktheriya, njl.njl., ukukhetha indlela efanelekileyo yokucoca kubalulekile.Indlela yokucoca i-centrifugal yekholomu inesantya sokutsalwa ngokukhawuleza kwaye inokususa ngempumelelo ukungcola okuchaphazela ukusabela okulandelayo kwe-enzymatic ye-RNA, kodwa iyabiza (i-Forgene inokubonelela ngezixhobo ezinexabiso eliphantsi, iinkcukacha ezithe vetshe cofa.Apha);usebenzisa iindlela zokucocwa kwezoqoqosho kunye neklasikhi, ezifana nemvula ye-LiCl, inokufumana iziphumo ezanelisayo, kodwa ixesha lokusebenza lide..

"Iindlela zokuziphatha ezintathu kunye neeNgqwalasela ezisibhozo" kwi-RNA Extraction

Uqeqesho 1:Ukuphelisa ukungcoliseka kwee-exogenous enzymes.

Qaphela 1:Nxiba ngokungqongqo iimaski kunye neiglavu.

Inqaku lesi-2:Iityhubhu ze-centrifuge, ii-Tip heads, iintonga ze-pipette, iitanki ze-electrophoresis, kunye neebhentshi zokulinga ezibandakanyekayo kuvavanyo kufuneka zilahlwe ngokucokisekileyo.

Inqaku lesi-3:Iirejenti/izisombululo ezibandakanyekayo kuvavanyo, ngakumbi amanzi, kufuneka zingabina RNase.

Uqeqesho 2:Vimba umsebenzi we-endogenous enzymes

Inqaku lesi-4:Khetha indlela efanelekileyo ye-homogenization.

Inqaku lesi-5:Khetha i-lysate efanelekileyo.

Inqaku lesi-6:Lawula isixa sokuqala sesampulu.

Uqeqesho 3:Cacisa injongo yakho yokutsalwa

Inqaku lesi-7:Ngayo nayiphi na inkqubo ye-lysate esondela kwisixa esikhulu sokuqala sesampulu, izinga lempumelelo yokukhutshwa lihla kakhulu.

Inqaku lesi-8:Ekuphela kwenqobo yokulinganisa yezoqoqosho yokutsalwa kwe-RNA eyimpumelelo yimpumelelo kwimifuniselo elandelayo, hayi isivuno.

Imithombo ye-10 ephezulu yokuNgcola kwe-RNase

1. Iminwe ngumthombo wokuqala we-exogenous enzymes, ngoko ke iiglavu kufuneka zinxitywe kwaye zitshintshwe rhoqo.Ukongeza, iimaski kufuneka zinxitywe, kuba ukuphefumla kukwangumthombo obalulekileyo wee-enzymes.Inzuzo eyongezelelweyo yokunxiba imaski yeglavu kukukhusela umfuni.

2. Iingcebiso ze-Pipette, ii-tubes ze-centrifuge, ii-pipettes - i-RNase ayinakunyanzeliswa nge-sterilization yodwa, ngoko iingcebiso ze-pipette kunye neebhubhu ze-centrifuge kufuneka ziphathwe nge-DEPC, nokuba ziphawulwe njenge-DEPC ephathwayo.Kungcono ukusebenzisa i-pipette yenjongo ekhethekileyo, uyisule nge-75% yebhola yekotoni yotywala ngaphambi kokusetyenziswa, ngakumbi intonga;ukongeza, qiniseka ukuba ungasebenzisi intloko yokususa.

3. Amanzi/isithinteli masingabinasulelo lwe-RNase.

4. Ubuncinci itafile yokuvavanya kufuneka ihlanjululwe nge-75% yeebhola zekotoni zotywala.

I-5.Endogenous RNase Zonke izicubu ziqulethe i-enzymes engapheliyo, ngoko ke ukukhenkceza ngokukhawuleza kwezicubu kunye ne-nitrogen engamanzi yindlela efanelekileyo yokunciphisa ukuthotywa.Indlela yokugcinwa kwe-nitrogen engamanzi / indlela yokugaya ngokwenene ayilunganga, kodwa yindlela yodwa yezicubu ezinamazinga aphezulu e-endogenous enzymes.

6. Iisampulu ze-RNA Iimveliso zokutsalwa kwe-RNA zinokuqulatha ungcoliseko lwe-RNase.

7. I-Plasmid extraction I-Plasmid extraction ihlala isebenzisa i-Rnase ukuthobisa i-RNA, kwaye i-Rnase eseleyo kufuneka igaywe ngeProteinase K kwaye ikhutshwe yi-PCI.

8. Ugcino lwe-RNA Nokuba igcinwe kwiqondo lobushushu eliphantsi, umkhondo wexabiso le-RNAse liya kubangela ukuthotywa kwe-RNA.Isisombululo esilungileyo sokugcinwa kwexesha elide le-RNA yityuwa / ukumiswa kotywala, kuba utywala luvimbela yonke imisebenzi ye-enzymatic kumaqondo aphantsi.

9. Xa i-cations (Ca, Mg) iqulethe le ion, ukufudumeza kwi-80C imizuzu emi-5 kuya kubangela ukuba i-RNA ihlulwe, ngoko ke ukuba i-RNA idinga ukufudumeza, isisombululo sokugcina kufuneka sibe ne-agent chelating (1mM Sodium Citrate, pH 6.4).

10. Ii-Enzymes ezisetyenziswe kwimifuniselo elandelayo zinokungcoliswa yi-RNase.

Iingcebiso ezili-10 zokutsalwa kwe-RNA

I-1: Ukuthintela ngokukhawuleza umsebenzi we-RNase.Iisampulu zikhenkcezwa ngokukhawuleza emva kokuqokelela, kwaye i-RNase ayisebenzi ngokusebenza ngokukhawuleza ngexesha le-lysis.

I-2: Khetha indlela efanelekileyo yokukhutshwa kwezicubu ezinomxholo ophezulu we-ribozyme, kunye nezicubu ze-adipose zilungele ukusebenzisa indlela equkethe i-phenol.

I-3: Umgangatho wokubikezela ufuna uMntla, ukwakhiwa kwethala leencwadi le-cDNA kufuna ingqibelelo ephezulu, kwaye i-RT-PCR kunye ne-RPA (i-Ribonuclease protection assay) ayifuni ingqibelelo ephezulu.I-RT-PCR idinga ukucoceka okuphezulu (iintsalela ze-enzyme inhibitor).

I-4: I-homogenization ngokucokisekileyo iyona nto iphambili ekuphuculeni isivuno kunye nokunciphisa ukuthotywa.

I-5: Khangela ingqibelelo ye-RNA electrophoresis ukufumanisa, i-28S: 18S = 2: i-1 isibonakaliso esipheleleyo, i-1: i-1 iyamkeleka kwiimvavanyo ezininzi.

I-6: Ukususwa kwe-DNA ye-RT-PCR, uhlalutyo lwe-array Kungcono ukusebenzisa i-Dnase I ukususa i-DNA.

I-7: Ukunciphisa ukungcoliswa kwee-enzymes zangaphandle - i-enzyme ayinakungeniswa ngaphandle.

I-8: Xa ugxininise i-nucleic acid ephantsi, i-reagent co-precipitation kufuneka yongezwe.Kodwa ukuthintela i-co-precipitant equkethe i-enzymes kunye nokungcoliswa kwe-DNA.

I-9: Ukunyibilikisa i-RNA, ukuba kuyimfuneko, ukushisa kwi-65C imizuzu emi-5.

indlela yokugcina efanelekileyo

Inokugcinwa kwi--20C ixesha elifutshane, kwaye kwi -80C ixesha elide.Isinyathelo sokuqala ekuphuculeni izivuno ze-RNA kukuqonda ukuba umxholo we-RNA weesampuli ezahlukeneyo uyahluka kakhulu.Ubuninzi obuphezulu (2-4ug / mg) njengesibindi, i-pancreas, intliziyo, ubuninzi obuphakathi (0.05-2ug / mg) njengengqondo, umbungu, intso, imiphunga, i-thymus, i-ovary, i-low abundance (<0.05ug / mg) mg) njenge-bladder, ithambo, amafutha.

I-1: Iiseli ze-Lyse ukukhulula i-RN - ukuba i-RNA ayikhululwa, isivuno siya kuncitshiswa.I-homogenization yombane isebenza ngcono kunezinye iindlela ze-homogenization, kodwa kusenokufuneka zidityaniswe nezinye iindlela, ezinje ngolwelo lwenitrogen yokuxutywa, ukugaywa kwe-enzymatic (Lysozyme/Lyticase)

2: Ukuphucula indlela yokukhupha.Iingxaki ezinkulu kwiindlela ezisekelwe kwi-phenol ziyi-stratification engaphelelanga kunye nokulahlekelwa kwe-RNA inxalenye (i-supernatant ayikwazi ukususwa ngokupheleleyo).I-stratification engaphelelanga ngenxa ye-nucleic acid ephezulu kunye nomxholo weprotheni, onokusombululwa ngokunyusa inani le-lysate esetyenzisiweyo okanye ukunciphisa inani lesampulu.Inyathelo lokutsalwa kweklorofomu yongezwa kwizicubu ze-adipose.Ilahleko ye-RNA inokuncitshiswa ngokumpompa ngasemva okanye ngokususa i-organic layer ilandelwa yi-centrifugation.Eyona ngxaki inkulu kunye neendlela ezisekelwe kwikholamu ye-centrifugation yisampuli engaphezulu.

Iingcebiso zokutsalwa kweClassic

1. Ukuhlanjululwa kwePhenol: Yongeza umthamo olinganayo we-1: 1 Phenol / Chloroform kwaye udibanise ngamandla kwi-1-2 imizuzu.I-Centrifuge ngesantya esiphezulu kwimizuzu emi-2.Susa ngononophelo i-supernatant (80-90%).Ungaze ufike kumaleko aphakathi.Umthamo olinganayo wesisombululo sokusabela unokongezwa kwiPhenol / Chloroform kunye ne-supernatant isuswe.Ezi zimbini ze-supernatants zinokuxutywa kunye kwi-nucleic acid precipitation ukuphucula isivuno.Musa ukuba mnene kakhulu xa uxuba, kwaye ungazami ukususa yonke i-supernatant.

2. Ukuhlamba nge-ethanol ye-70-80%: Ngexesha lokuhlamba, i-nucleic acid kufuneka inqunyanyiswe ukuqinisekisa ukuba ityuwa eseleyo ihlanjwe.Ngelo xesha, ngokukhawuleza emva kokuthulula i-ethanol, i-centrifuge ngesantya esiphezulu imizuzwana embalwa, uze ususe i-ethanol eseleyo ngepipette.Ukunyibilika emva kokuma kwindawo yokushisa kwe-5-10 imizuzu.

11. Ukukhutshwa kwemibutho ekhethekileyo

1. Izicubu ze-Fibrous: Isitshixo kwi-RNA yokukhutshwa kwi-fibrous tissue ezifana nentliziyo / i-skeletal muscle kukuphazamisa ngokupheleleyo izicubu.Ezi zicubu zinokuxinwa kweeseli eziphantsi, ngoko ke ubungakanani be-RNA ngeyunithi yobunzima beethishu buphantsi, kwaye kungcono ukusebenzisa isixa sokuqala kangangoko kunokwenzeka.Qinisekisa ukuba usila izicubu ngokucokisekileyo phantsi kweemeko zokukhenkceza.

2. Izicubu ezineprotheyini ephezulu / i-fat content: umxholo wengqondo / wemifuno uphezulu.Emva kokukhutshwa kwe-PCI, i-supernatant iqulethe iifloccules ezimhlophe.I-supernatant kufuneka iphinde ikhutshwe nge-chloroform.

3. Izicubu ezinomxholo ophezulu we-nucleic acid / ribozyme: i-spleen / thymus ine-nucleic acid ephezulu kunye nomxholo we-ribozyme.Ukugaya izicubu phantsi kweemeko zokukhenkceza ezilandelwa yi-homogenization ekhawulezileyo inokusebenza ngokufanelekileyo i-ribozymes.Nangona kunjalo, ukuba i-lysate i-viscous kakhulu (ngenxa yomxholo ophezulu we-nucleic acid), ukukhutshwa kwe-PCI akuyi kukwazi ukwenza i-stratify ngempumelelo;Ukongeza i-lysate ngakumbi kunokusombulula lo mba.Ukutsalwa kwe-PCI ezininzi kunokususa i-DNA eyintsalela.Ukuba i-white precipitate ifom ngokukhawuleza emva kokongeza utywala, ibonisa ukungcoliswa kwe-DNA.Ukukhutshwa kwakhona nge-PCI ene-acidic emva kokuchithwa kunokususa ukungcoliswa kwe-DNA.

4. Izicubu zezityalo: Izicubu zezityalo zintsonkothile ngakumbi kunezicubu zezilwanyana.Ngokuqhelekileyo, izityalo zigutyungelwa phantsi kweemeko zenitrogen yolwelo, ngoko ke ukuthotywa kwe-RNA yi-endogenous enzymes ayiqhelekanga.Ukuba ingxaki yokuthotywa ayisonjululwanga, iphantse yabangelwa bubumdaka obuqulethwe kwisampulu.Ukungcola okuqulethwe kwizityalo ezininzi kuya kukhokelela kwiintsalela, kwaye isizathu sokuba iintsalela kaninzi kungenxa yokuba obu kungcola kunokufana okuthile kunye ne-RNA: uyanyuka kwaye ndiya kugalela, kwaye uyabhengeza kwaye ndiyabhengeza.Ezi mpawu zigqiba ukuba zinamandla kakhulu e-enzyme inhibitors.

Okwangoku, ii-reagents ze-RNA zokurhweba ziyakwazi ukulungelelaniswa phantse kuzo zonke izicubu zezilwanyana ezinohlengahlengiso oluncinci, kodwa zimbalwa ii-reagents zokurhweba ze-RNA ezinokuthi zilungele izicubu ezininzi zezityalo.Ngethamsanqa, i-Foregene inokubonelela ngokukhethekileyoiikhithi zokukhutshwa kwe-RNA, siPlant Total RNA Isolation kit, Plant Total RNA Isolation kit Plus.Le yokugqibela yenzelwe ngokukhethekileyo izityalo ezinepolysaccharide ephezulu kunye nomxholo wepolyphenol.Ngokutsalwa kwe-RNA, ingxelo evela kubasebenzisi baselebhu ilunge kakhulu.

12. Iziphumo zesampulu yokukhenkceza kunye nokunyibilika Isampulu engumkhenkce inokuba nkulu, kwaye kufuneka isikwe phambi kokuba isetyenziselwe ukutsalwa kwe-RNA.Iisampuli zivame ukunyibilika (mhlawumbi inxalenye) ngexesha lokusika.Iisampulu ezikhenkcezisiweyo zinokufuneka zilinganiswe phambi kokutsalwa kwe-RNA, kwaye ukunyibilika kuya kwenzeka ngokuqinisekileyo ngexesha lale nkqubo.Ngamanye amaxesha, ukunyibilika kwesampulu kwenzeka ngexesha lenkqubo yolwelo nitrogen yokusila;okanye isampulu ekhenkcezisiweyo yongezwa ngokuthe ngqo kwi-lysate ngaphandle kokusila i-nitrogen engamanzi, kwaye ukunyibilika kuya kwenzeka ngokuqinisekileyo phambi kokuba i-homogenization epheleleyo.Iimvavanyo zibonise ukuba izicubu ezinomkhenkce zithandeka ngakumbi ekonakaleni kwe-RNA ngexesha lokunyibilika kunezicubu ezitsha.Esona sizathu: Inkqubo yokunyibilika komkhenkce iphazamisa izakhiwo ezingaphakathi kwiseli, yenze kube lula ukuba ii-endogenous enzymes zidibane ngqo ne-RNA.

13. Isigwebo somgangatho we-RNA Ngokuqhelekileyo, i-electrophoresis isetyenziselwa ukugweba ingqibelelo ye-RNA, kwaye i-A260 / A280 isetyenziselwa ukugweba ukucoceka kwe-RNA.Kwithiyori, i-RNA ecocekileyo inomlinganiselo we-28S: 18S = 2.7: 1, kwaye idatha eninzi igxininisa umlinganiselo we-28S: 18S = 2: 1.Inyaniso kukuba phantse akukho nanye ye-RNA ekhutshwe kwiisampuli ngaphandle kweeseli kwi-2: umlinganiselo we-1 (oku kufunyenwe ngokusebenzisa i-Agilent Bioanalyzer).

Iziphumo ze-electrophoresis ze-RNA zichatshazelwa yizinto ezininzi, kubandakanywa isakhiwo sesibini, iimeko ze-electrophoresis, umthwalo wesampuli, i-degree of saturation yi-EB, njl.Ukuba i-28S kwi-2kb kunye ne-18S kwi-0.9kb icacile, kunye ne-28S: 18S> 1, ingqibelelo inokuhlangabezana neemfuno zeemvavanyo ezininzi ezilandelayo.

I-A260/A280 sisilathisi esibangele ukubhideka okukhulu.Okokuqala, kuyimfuneko ukucacisa intsingiselo yokuqala yesi salathisi se-nucleic acids: i-RNA ecocekileyo, i-A260 / 280 yayo = malunga ne-2.0.I-RNA ecocekileyo 'yisizathu' kunye ne-A260/A280 = 2 'yisiphumo'.Ngoku wonke umntu usebenzisa i-A260/A280 'njengesizathu', ecinga ukuba "ukuba i-A260/A280 = 2, ngoko ke i-RNA imsulwa", nto leyo ekhokelela ngokwemvelo ekubhidekeni.

Ukuba unomdla, unokongeza i-reagent encinci ehlala isetyenziselwa ukukhutshwa, njenge-phenol, i-guanidine isothiocyanate, i-PEG, njl., kwisampuli yakho ye-RNA, kwaye emva koko ulinganise umlinganiselo we-A260 / A280.Inyani yeyokuba uninzi lwezixhobo ezisetyenziselwa ukutsalwa kwe-RNA, kunye nokungcola okuninzi kwisampulu, zifunxa malunga ne-A260 kunye ne-A280, echaphazela i-A260/A280.

Eyona ndlela ifundisayo ngoku kukuskena iisampuli ze-RNA kuluhlu lwe-200-300 nm.Ijika le-RNA ecocekileyo inezi mpawu zilandelayo: i-curve igudile, i-A230 kunye ne-A260 ngamanqaku amabini okuguqa, i-A300 isondele kwi-0, i-A260 / A280 = malunga ne-2.0, kunye ne-A260 / A230 = malunga ne-2.0.Ukuba idatha yokuskena ayifumaneki, umlinganiselo we-A260/A230 nawo kufuneka umiselwe, njengoko lo mlinganiso unovakalelo ngakumbi kwi-carryover yabo bonke ukungcola okuchaphazela ukusabela kwe-enzymatic.Thatha ingqalelo uluhlu lomgca wesixhobo (0.1–0.5 ye-A260).

Kukho ezinye izinto ezimbini eziluncedo: umlinganiselo uya kuba malunga ne-0.3 ngaphantsi xa i-A260 / A280 ilinganiswa emanzini;ngelixa umlinganiselo olinganiswe kwi-10 mM EDTA imalunga ne-0.2 ephezulu kunomlinganiselo we-1 mM EDTA.

Iimveliso eziyeleleneyo:

China Plant Total RNA Isolation Kit Manufacturer kunye noMboneleli |I-Foregene (foreivd.com)

Uluhlu lokuzihlukanisa lwe-RNA Ababoneleli kunye neFactory |Uthotho lwe-RNA yodwa yaseTshayina Abavelisi (foreivd.com)

Uluhlu lokuzahlula lwe-RNA-Foregene Co., Ltd. (foreivd.com)