Intshayelelo:

I-RNA, igama lenzululwazi i-ribonucleic acid, isifinyezo sayo sivela kwi-RiboNucleic Acid, luhlobo lwe-nucleic acid eyenziwe ngokudibanisa ubuncinane be-ribonucleotides ngeebhondi ze-phosphodiester.

Isiseko sokuhlola i-RNA kukukhupha i-RNA engangcolisekanga, kwaye umgaqo osisiseko wokutsalwa kwe-RNA unokushwankathelwa ngolu hlobo lulandelayo:

1. Ukuphazamisa iiseli kuqala

2. Emva koko susa ukungcola kwe-macromolecular (iiprotheni, i-lipids, i-DNA, njl.)

3. Ekugqibeleni, i-RNA iyancipha ngelixa isusa iityuwa kunye nezinyibilikisi eziphilayo, kwaye ihlambulula ngakumbi i-RNA.

Ixesha elidlulileyo nelikhoyo lendlela yokukhupha

Isizukulwana sokuqala sendlela yokukhutshwa kwe-RNA yindlela ye-TRIzol yemvula (ii-reagents ezisetyenzisiweyo ziquka i-guanidine isothiocyanate, i-phenol, i-chloroform, njl.) eyaqanjwa nguyise omkhulu ogama linguPiotr Chomczynski kunye nogogo uNicoletta Sacchi, kuba le nto yokuvelisa ivumela ukukhutshwa kwe-RNA ngokukhawuleza kwaye kulula, ukususela ngoko waqala uphando lwe-RNA.

Kamva, uTatomkhulu wathengisa le patent kwi-Invitrogen, kwaye i-Invitrogen yakhuthaza le ndlela kwihlabathi kwaye yaba yindlela esetyenziswa ngokubanzi.

Kamva, i-Invitrogen yafunyanwa nguThermo!

Imveliso emele: i-reagent ye-TRIzol ye-Invitrogen

Nangona kunjalo, indlela ye-TRIzol yemvula idinga imvula eninzi kunye nokukhutshwa kwe-RNA, kwaye i-reagents esetyenzisiweyo inetyhefu kwaye ithatha ixesha elide ukusebenza.

Ke ngoko, indlela yokucoca i-enzyme yesizukulwana sesibini yabakho, kusetyenziswa i-silica gel membrane njengesixhobo esithile se-nucleic acid adsorption ukukhulisa ukubuyiswa kwe-RNA kwisampulu ngelixa kususwa obunye ukungcola.

Indlela yesizukulwana sesibini yenza lula ukusebenza ngesiseko sokusebenza okuzinzile, ukunciphisa umngcipheko, kunye nokuphucula ukusebenza kakuhle kwe-RNA extraction.

Iimveliso ezimele: QIAGEN RNeasy kunye neemveliso ezininzi zasekhaya

Nangona kunjalo, zombini iindlela zesizukulwana sokuqala kunye nesibini zifuna ukusetyenziswa kwe-DNase ukususa ukungcoliseka kwe-DNA, okuzisa ukuphazamiseka kwiimvavanyo ezilandelayo ezisezantsi.I-DNase eshiyekileyo iya kunciphisa ukusebenza kakuhle kokubhalwa kwe-reverse kunye nokunciphisa i-cDNA synthesis ye-strand yokuqala, ngaloo ndlela ichaphazela ukuchaneka kweziphumo.

Ngoko ke, indlela yokukhutshwa kwesizukulwana sesithathu iphuculwe ngesiseko sesizukulwana sesibini.Uvavanyo aludingi ukongeza naziphi na ii-enzymes, kwaye i-RNA inokufumaneka ngokulula ngeekholamu ezimbini kuphela.

Ikholamu 1: IKholam yokucoca iDNA yokucoca iDNA

UKholam 2: Umhlathi we-RNA kuphela ubhengeza kwaye uphinde ufumane i-RNA

Umsebenzi olula kangaka awugcini nje kuphela ixesha, kodwa uququzelela ukusebenza kovavanyo olusezantsi!

Iimveliso ezimele: QIAGEN RNeasy Plus kunyeI-ForegeneIimveliso zokuhlanjululwa kwe-RNA

(Neasy Plus)

(I-Foregene'sIsiTyalo Sisonke seKit yeRNA)

Ndixelele ngokuthula!Ikhithi ye-RNA ye-Foregene ikwabonisa i-RNase Eraser ekulula ukuyisebenzisa, enokususa ngokupheleleyo i-400μg ye-RNase kwindawo eqinileyo ngomzuzu omnye, ngaphandle kokongeza naziphi na izinto eziyityhefu emzimbeni womntu!

Pic1: Itshathi yokuthelekisa inkqubo yokusebenza

Iimveliso ze-Foregene zinokugqiba ukutsalwa kwe-RNA kwiingoma nje ezimbini, musa ukwenza abanye babe nomona ngenkqubela yakho yovavanyo!

Pic2:Eiziphumo zovavanyo itshathi yokuthelekisa

Isizukulwana sokuqala (1: TRIzol)

Isizukulwana sesibini (2: RNeasy of QIAGEN)

Isizukulwana sesithathu (3: QIAGEN's RNeasy plus, 4: Foregene)

Pic3: igrafu ye-qPCR (igrafu yokukhulisa igophe)

Isizukulwana sokuqala (okuluhlaza kuthetha i-TRIzol -, ebomvu ithetha i-TRIzol +)

Isizukulwana sesibini (isibhakabhaka esiluhlaza sithetha QIAGEN RNeasy-, obomvu uthetha QIAGEN RNeasy +)

Isizukulwana sesithathu (iblue ithetha QIAGEN RNeasy plus, eluhlaza ithetha Forgenene)

(Qaphela: igrafu ye-qPCR "-" ibonisa ukuba i-DNase ayizange yongezwe kwaye ukungcoliswa kwe-DNA akuzange kususwe. Ngoko ke, i-CT iphezulu kwaye umphumo awuchanekanga; "+" ibonisa ukuba i-DNase yongezwa kwaye ukungcola kwe-DNA kususiwe, umphumo uchanekile)

Lonke uphuculo lobugcisa luthetha ukuba ukusebenza kovavanyo luphuhla ngendlela elula nelula ngakumbi yomsebenzisi.

I-Foregene ijolise ekuncedeni abaphandi abaninzi bezenzululwazi bonge ixesha lovavanyo kunye neendleko, ukufumana iziphumo zovavanyo olukumgangatho ophezulu, kunye nokudala ngokudibeneyo impumelelo yophando lwenzululwazi ebaluleke ngakumbi.