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1. Khangela ukufunxwa kwesisombululo se-RNA

I-Absorbance kwi-280, i-320, i-230, kunye ne-260 nm imele amaxabiso e-nucleic acid, imvelaphi (i-solution turbidity), i-concentration yetyuwa, kunye nezinto eziphilayo ezifana neprotheni, ngokulandelanayo.Ngokuqhelekileyo jonga kuphela kwi-OD260/OD280 (Umlinganiselo, R).Xa i-1.8 ~ 2.0, sicinga ukuba ukungcoliswa kweprotheyini okanye enye into ephilayo kwi-RNA inokunyamezela, kodwa kufuneka kuqatshelwe ukuba xa i-Tris isetyenziswe njenge-buffer ukuze ibone ukuxhamla, ixabiso le-R linokuba likhulu kune-2 (ngokubanzi kufuneka ibe <2.2).Xa i-R <1.8, ukungcoliswa kweprotheyini okanye enye into ephilayo kwisisombululo ibonakala ngokucacileyo, kwaye i-fate ye-RNA inokumiselwa ngokweemfuno.Xa i-R>2.2, ithetha ukuba i-RNA iye yafakwa i-hydrolyzed ibe yi-nucleic acid enye.
 
2.Ipateni ye-Electrophoretic ye-RNA
Ngokuqhelekileyo, ijeli ye-denaturing isetyenziselwa i-RNA electrophoresis, kodwa ukuba yenzelwe kuphela ukufumanisa umgangatho we-RNA, ijeli ye-denaturing ayiyimfuneko, kwaye ijeli ye-agarose eqhelekileyo ingasetyenziswa.Injongo ye-electrophoresis kukubona ukunyaniseka kweebhendi ze-28S kunye ne-18S kunye nomlinganiselo wazo, okanye ukunyaniseka kwe-mRNA smear.Ngokuqhelekileyo, ukuba iibhendi ze-28S kunye ne-18S zikhanya, zicacile, kwaye zibukhali (ngokubhekiselele kwimida yeebhendi zicacile), kunye nokukhanya kwe-28S kuphindwe kabini kune-18S band, sicinga ukuba umgangatho we-RNA ulungile.
Ezi zingasentla ziindlela ezimbini esiqhele ukuzisebenzisa, kodwa akukho nanye kwezi ndlela zimbini inokusixelela ngokucacileyo ukuba kukho intsalela ye-RNase kwisisombululo se-RNA.Ukuba kukho inani elincinane kakhulu le-RNase kwisisombululo, kunzima ngathi ukuyibona ngale ndlela ingentla, kodwa uninzi lweempendulo ze-enzymatic ezilandelayo zenziwa ngaphezu kwama-37 degrees kunye nexesha elide.Ngale ndlela, ukuba kukho inani elincinci le-RNase kwisisombululo se-RNA, ngoko kuya kubakho indawo efanelekileyo kakhulu kunye nexesha lokudlala indima yabo kwiimvavanyo ezilandelayo, kwaye ngokuqinisekileyo uvavanyo luya kubanda ngeli xesha.Ngezantsi sazisa indlela enokuqinisekisa ukuba kukho i-RNase eshiyekileyo kwisisombululo se-RNA.
 
3. Uvavanyo lokugcinwa kobushushu
Ngokutsho kwesampulu yoxinaniso, zoba ezimbini ze-1000 ng RNA kwisisombululo se-RNA kwaye udibanise kwi-tube ye-0.5 ml ye-centrifuge, kwaye uyifake kunye ne-pH 7.0 Tris buffer kumthamo opheleleyo we-10 ul, kwaye emva koko utywine i-cap ye-tube.Beka enye yazo kwindawo yokuhlambela amanzi kwiqondo lokushisa elingaguqukiyo kwi-70 ° C kwaye uyigcine ishushu iyure eli-1.Enye inxalenye yayigcinwe kwi -20 ° C efrijini ngeyure eli-1.Xa ixesha liphelile, susa iisampuli ezimbini ze-electrophoresis.Emva kokuba i-electrophoresis igqityiwe, thelekisa iibhendi ze-electrophoretic ezimbini.Ukuba iibhanti zezi zibini zihambelana okanye azikho umehluko omkhulu (ewe, amaqela abo nawo adibana neemeko kwindlela ye-2), kuthetha ukuba akukho ngcoliseko ye-RNase eseleyo kwisisombululo se-RNA, kwaye umgangatho we-RNA ulungile kakhulu.Ngokuchasene noko, ukuba isampuli efakwe kwi-70 ° C ibonisa ukuthotywa okucacileyo, kubonisa ukuba kukho ukungcola kwe-RNase kwisisombululo se-RNA.
 
2 Iindlela zovavanyo kunye nobuchule bokutsalwa kwe-RNA
Iingxaki esihlala sidibana nazo xa sikhupha iRNA zezi: (1) Isivuno seRNA siphantsi;(2) I-RNA inongcoliso olunzulu lwetyuwa;(3) I-RNA inongcoliseko olunzulu lwezinyibilikisi eziphilayo;(4) ukuthotywa kwesampuli kunye nezinye iingxaki
 
1. Ngokuqhelekileyo zisetyenziswa ngokuqhelekileyo i-reagents yokukhupha i-RNA
Indlela ye-guanidine isothiocyanate kunye nendlela ye-Trizol yizona ndlela ezisetyenziswa ngokuqhelekileyo zokukhutshwa kwe-RNA iyonke kwizicubu zezilwanyana kunye neeseli zezilwanyana.Kufanelekile ngokukodwa kwiisampuli ezincinci kunye nezicubu ezinzima kakhulu ukukhupha, ezifana nokutsalwa kwe-RNA iyonke kwisikhumba somvundla kunye nezicubu ezixhamlayo zezilwanyana;Ukongeza, i-Trizol, njenge-reagent yenjongo jikelele ye-lysis, ingasetyenziselwa ukukhutshwa kwezicubu zezityalo, iibhaktheriya, i-fungi kunye nezinye izicubu.Kwizicubu zezityalo eziqulethe i-polysaccharides kunye ne-polyphenols, njenge-camellia oleifera, amaqabunga eti, i-rapeseed, njl., Indlela ye-CTAB ingasetyenziselwa ukukhupha i-RNA epheleleyo.

Njengendlela eqhelekileyo, indlela yekholamu ephindwe kabini nayo ithandwa kakhulu ngenxa yokusebenza kwayo kweqondo lokushisa eliqhelekileyo, akukho mfuneko yokongeza i-RNase, kunye nokhuseleko-akukho chloroform, i-phenols kunye nezinye i-organic reagents zokukhutshwa.(iimveliso ezicetyiswayo )

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2. Ukukhutshwa kwe-RNA iyonke kwiithishu zezilwanyana
 
(1) Zama ukukhetha izicubu ezisandul’ ukutsha, ukuba azintshanga (ngokukhethekileyo kwisithuba seenyanga ezintathu – 80 ℃ isikhenkcezisi okanye umkhenkce kwinitrogen engamanzi.” Xa usika inyama, musa ukusika ngokuthe ngqo kwiqondo lobushushu legumbi, qiniseka ukuba uyibeke kwibhokisi yomkhenkce, zama ukukuphepha ukukhenkceza ngokuphindaphindiweyo nokunyibilika.
(2) Sebenzisa isikere esicocekileyo kunye neetweezers ukusika iqhekeza elincinci lethishu, zama ukusika indawo esembindini yethishu xa usika isampulu, okanye uqale usike iqhekeza elikhulu lethishu phakathi, uze usike isampuli kwindawo entsha yokusikwa.Izicubu ezisusiweyo kufuneka zihlanjululwe ngokupheleleyo, zifake i-tissue edibeneyo kwi-tube ye-EP ngaphandle kwe-RNase, yongeza i-lysate, i-tissue edibeneyo kufuneka ibonakaliswe ngokupheleleyo kwi-lysate, kwaye ilungiselele i-homogenization.

(3) Kwizicubu eziqhelekileyo, khetha izicubu ezinobungakanani beembotyi ze-mung (30-60 mg) kwi-homogenization.Ukuba izicubu ziqulethe inani elikhulu leprotheyini, i-fat, okanye i-fibrous tissues exineneyo njengesibindi, ngokufanelekileyo ukwandisa okanye ukunciphisa inani lezicubu ezisikiweyo (ukhetho) Khetha i-10 ~ 20 mg).
(4) Ukuba i-fish muscle, inyama ye-shrimp, i-jellyfish kunye nezinye izicubu ezinomthamo ophezulu wamanzi zikhutshwe, umthamo wesampuli kufuneka unyuswe ngokufanelekileyo (kucetyiswa i-100-200 mg).
(5) Ukuba iimeko zivuma, isihlunu sesilwanyana sinokutsalwa ngokuthe ngqo emva kokuba senziwe homogenizer yethishu ekwipaseji ephezulu, ukuba asikho isixhobo esinjalo.
(6) I-RNA efunyenwe emva kokukhutshwa kokugqibela kufuneka ifakwe kwibhokisi lomkhenkce ngokukhawuleza ukunciphisa ukuthotywa kwe-RNA.

3. Iseli yezilwanyana iRNA extraction

(1) Iiseli zokumisa: i-centrifuge ngokuthe ngqo kwaye ulahle i-medium, uhlambe nge-PBS eyinyumba ngamaxesha angama-1-2, uze umise ngexabiso elifanelekileyo le-PBS, uze ungeze i-lysate ye-lysis.Musa ukongeza i-lysate ngokuthe ngqo kwiiseli ze-precipitated emva kokulahla ngokupheleleyo ulwelo.Oku kuya kubangela ukuba ipakethe ye-histone ekhutshwe emva kweeseli ze-lysed kumaleko angaphandle ukuba ibambelele ngaphandle kweeseli ezimanzi, ngaloo ndlela inciphisa uqhagamshelwano lweeseli ngaphakathi kwepellet kunye ne-lysate., okubangela ukuba i-cell lysis ingaphelelanga kunye nokunciphisa isivuno se-RNA.

(2) Iiseli ezihambelanayo okanye ezingabambeleli ngokuqinileyo: Emva kokulahla i-medium, hlamba kunye ne-PBS ngamaxesha angama-1-2, emva koko ufunxe ngokuthe ngqo inani elifanelekileyo le-PBS kwaye uvuthele isitya senkcubeko kunye ne-pipette okanye umpu ukuvuthela iiseli, kwaye uzidlulisele kwiiseli ze-RNA-free.Yongeza i-lysate kwi-tube ye-EP ye-enzyme yokukhutshwa.

(3) Iiseli ezibambelelayo: kufuneka zigaywe nge-trypsin kuqala, emva koko ziqokelelwe kwiityhubhu ze-EP ze-RNase-free, i-centrifuged ukususa i-supernatant, ihlanjwe ngamaxesha angama-1-2 nge-PBS ukususa i-trypsin engaphezulu, kwaye iphinde imiswe ngexabiso elifanelekileyo le-PBS Emva koko uqhubekele kwinqanaba lokukhupha.

4. Ukutsalwa kwe-RNA yezityalo

Izicubu zezityalo zizityebi kwi-phenolic compounds, okanye zizityebi kwi-polysaccharides, okanye ziqulethe i-metabolites yesibini engaziwayo, okanye inomsebenzi ophezulu we-RNase.Ezi zinto zidibene ngokuqinileyo kunye ne-RNA emva kwe-cell lysis ukwenza ii-complexes ezingaxutywanga okanye i-colloidal precipitates, okunzima ukuyisusa.Ngoko ke, xa sikhupha izicubu zezityalo, kufuneka sikhethe ikiti yezityalo.I-lysate kwikiti inokusombulula ngempumelelo iingxaki ze-oxidation elula ye-polyphenols kunye nokuhlukana kwee-polysaccharide compounds kunye ne-nucleic acids.

(Kwi-polysaccharide polyphenol plant RNA extraction, iimveliso ezicetyiswayo:

(1) Ixolo, i-pulp, imbewu, amagqabi, njl.Ngethuba lenkqubo yokusila, i-nitrogen engamanzi kufuneka izaliswe kwakhona ngexesha ukuphepha ukunyibilika isampuli.Isampulu yomhlaba kufuneka yongezwe ngokukhawuleza kwi-lysate kwaye ishukunyiswe ukuphepha ukuthotywa kwe-RNA.

(2) Iisampulu ezityebileyo zefiber ezifana nerayisi kunye namagqabi engqolowa, inani lokutsalwa kufuneka lincitshiswe ngokufanelekileyo, ngaphandle koko ukugaya izicubu kunye ne-lysis ayiyi kugqitywa, okubangela isivuno esincinci se-RNA ekhutshwe.

(3) Kwizicubu zezityalo ezinomxholo ophezulu wamanzi, ezifana neziqhamo zegromegranate, isiqhamo se-watermelon, isiqhamo se-peach, njl., Ubungakanani besampula kufuneka bunyuswe ngokufanelekileyo (100-200 mg inokuzikhethela).

(4) Izicubu zezityalo, ezifana namagqabi esityalo, i-rhizomes, iziqhamo ezinzima kunye nezinye izinto zicetyiswa ngokubanzi ukuba zisebenzise i-nitrogen engamanzi ukuze udaka ngokucokisekileyo izithako kwi-mortar, kwaye emva koko uqhubele phambili kwinqanaba lokukhupha.I-homogenizers yezicubu eziqhelekileyo ayinakusebenza kwi-homogenizing izicubu zezityalo, kwaye azikhuthazwa ngokubanzi.

5. Izilumkiso zokutsalwa kwe-RNA

(1) Iisampulu zezicubu kufuneka zibe zintsha kangangoko ukuphepha ukukhenkceza okuphindaphindiweyo nokunyibilika.

(2) Ithishu ifanele icolwe ngokupheleleyo xa itsalwa, yaye umlinganiselo wenyama awufanele ube mncinane kakhulu, singasathethi ke ngokuba zininzi kakhulu.

(3) Ixesha elaneleyo le-incubation kufuneka linikezwe emva kokongeza i-lysate ukuze i-lyse ngokupheleleyo isampuli.

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(5) Xa uhlamba, ulwelo lokuhlamba kufuneka lungene ngokupheleleyo lujikeleze udonga lwetyhubhu ukuqinisekisa ukuhlamba ngokucokisekileyo.

(6) Indlela yokukhutshwa kwekholamu, ngaphezu kokukhupha ikholamu emva kokuhlamba, ikholamu ye-adsorption kufuneka ifakwe kwi-bench ecocekileyo kakhulu kwaye ivuthelwe imizuzu emi-5-10 ukuze ikhuphe ngokupheleleyo i-solvent ye-organic ukuya kome.

(7) Kwi-elution yokugqibela yendlela yekholomu, emva kokongeza amanzi e-DEPC, kufuneka ifakwe kwi-3-5 imizuzu, okanye i-DEPC yamanzi kufuneka ifudunyezwe kwi-60 ° C kwangaphambili ukwandisa isivuno se-lution.Kwindlela yendabuko ye-Trizol cleavage kunye ne-isopropanol precipitation method, i-RNA yokugqibela ichithwa kumanzi e-DEPC, ngoko ke kufuneka kunikwe ixesha elifanelekileyo lokuchithwa, kwaye i-bottom ye-tube ye-centrifuge kufuneka iqhutyelwe ngokuqhubekayo kunye ne-pipette tip.

3 Three Oonobangela kunye nezisombululo zoxinaniso RNA ephantsi / umgangatho ombi
 
1. Isivuno siphantsi kakhulu
Isampuli ekhutshiweyo iphantsi kakhulu, isixa esipheleleyo asanelanga, okanye isampuli ekhutshiweyo ininzi kwaye i-lysis ayigqibekanga;izicubu okanye iiseli zomgangatho ofanelekileyo kufuneka zisetyenziswe ukukhutshwa, unyango lwangaphambili lwesampuli kufuneka lwenziwe kakuhle, kwaye i-lysis kufuneka yanele.
 
2. Iintsalela zeGenome
Xa kutsalwa ngendlela ye-Trizol, xa i-supernatant ifunxwa kumaleko aphakathi emva kokubekwa, ukungcoliseka okunzulu kwe-genome kuya kubangelwa;ukhathalelo olongezelelweyo kufuneka luthathwe xa ubeka umaleko ukuphepha ukufunxa kumaleko aphakathi.Ukuba indlela yekholomu isetyenziselwa utsalo, ikiti equlethe i-DNase Ndingakhethelwa ukutsalwa.I-nucleic acid adsorbed kwi-membrane igaywe ngokuthe ngqo nge-DNase I, enokunciphisa kakhulu iintsalela ze-DNA.
 
3. Ukuthotywa kweRNA
Isenokuba ukuthotywa kwesampulu ekhutshiweyo ngokwayo, okanye ukuthotywa okubangelwa ngexesha lenkqubo yokukhutshwa;kangangoko kunokwenzeka, iisampulu ezintsha kufuneka zisetyenziselwe ukukhutshwa kwe-RNA, kwaye iisampulu eziqokelelweyo kufuneka zigcinwe kwi-nitrogen engamanzi okanye -80 ° C efrijini ngexesha, kwaye ukukhenkceza okuphindaphindiweyo kunye nokunyibilika kufuneka kuthintelwe.Iingcebiso ze-RNase / DNase zamahhala, iityhubhu ze-centrifuge kunye nezinye izinto kufuneka zisetyenziswe kwinkqubo yokukhutshwa kwe-RNA.Inkqubo yokutsalwa kufuneka ikhawuleze kangangoko.I-RNA ekhutshiweyo kufuneka ifakwe kwibhokisi yeqhwa kwaye igcinwe kwi -80 ngexesha.Ukuba i-RNA ekhutshiweyo idinga ukufunyanwa nge-gel electrophoresis, i-electrophoresis kufuneka yenziwe ngokukhawuleza emva kokukhutshwa, kwaye i-electrophoresis buffer kufuneka ithathelwe indawo entsha elungiselelwe.
 
4. Iintsalela zetyuwa kunye ne-organic solvent
I-reagents yokukhupha iqulethe i-phenol kunye ne-guanidine ityuwa, kwaye isisombululo sokuhlamba sine-ethanol.Ngethuba lenkqubo yokukhupha, i-lysate ayizange ifakwe ngokupheleleyo kwaye ilahlwe, kwaye isisombululo sokuhlamba asizange somiswe ngokupheleleyo.Iityuwa ezishiyekileyo kunye nezinyibilikisi ze-organic ziyingozi kwi-reverse transcription kunye ne-PCR.Iidigri ezihlukeneyo zokuthintela, ngoko i-lysate ye-tissue kufuneka isuswe ngokupheleleyo ngexesha lokukhutshwa, kwaye ukuhlamba kufuneka kube okwaneleyo ukwenzela ukuba iindonga ezijikelezayo zombhobho zihlanjwe.Ukongezelela, ityhubhu ikhutshiwe kwaye ivuthelwe yinyathelo eliyimfuneko, eliya kunciphisa ngakumbi intsalela yezinto eziphilayo.
 
Ngolwazi oluthe kratya malunga nokutsalwa kwe-RNA, nceda ulandele iwebhusayithi yethu:
www.foreivd.com ngolwazi olungakumbi.

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Ixesha lokuposa: Dec-01-2022