Selelekela:

RNA, lebitso la saense ribonucleic acid, khutsufatso ea lona e tsoa ho RiboNucleic Acid, ke mofuta oa nucleic acid e entsoeng ka ho hokahanya bonyane li-ribonucleotide tse ngata ka li-phosphodiester bond.

Sepheo sa ho hlahloba RNA ke ho ntša Total RNA e se nang tšilafalo, 'me molao-motheo oa ho ntšoa ha RNA o ka akaretsoa ka tsela e latelang:

1. Senya lisele pele

2. Ebe u tlosa litšila tsa macromolecular (liprotheine, lipids, DNA, joalo-joalo)

3. Qetellong, RNA e na le pula ha e ntse e ntša letsoai le metsoako ea lintho tse hlapollang, 'me e tsoela pele ho hloekisa RNA.

Mokhoa oa khale le oa hona joale oa ho hula

Moloko oa pele oa mokhoa oa ho ntša RNA ke mokhoa oa TRIzol precipitation (li-reagents tse sebelisoang li kenyelletsa guanidine isothiocyanate, phenol, chloroform, joalo-joalo) e qapiloeng ke ntate-moholo ea bitsoang Piotr Chomczynski le nkhono Nicoletta Sacchi, hobane tlhahiso ena e lumella ho ntšoa ha RNA Ka potlako le ka mokhoa o bonolo, ho tloha ka nako eo ho qala lipatlisiso tsa RNA.

Hamorao, Ntate-moholo o ile a rekisa patent ena ho Invitrogen, ’me Invitrogen e ile ea ntšetsa pele mokhoa ona lefatšeng ’me ea e-ba mokhoa o sebelisoang haholo.

Hamorao, Invitrogen e ile ea fumanoa ke Thermo!

Sehlahisoa sa moemeli: Reagent ea Invitrogen's TRIzol

Leha ho le joalo, mokhoa oa TRIzol oa pula o hloka pula e ngata le ho ntšoa ha RNA, 'me li-reagents tse sebelisoang li chefo' me li nka nako e telele ho sebetsa.

Ka hona, ho ile ha e-ba le mokhoa oa ho hloekisa oa enzyme ea moloko oa bobeli, o sebelisa lesela la silika la gel e le lisebelisoa tse khethehileng tsa nucleic acid adsorption ho eketsa ho hlaphoheloa ha RNA ka sampuli ha ho ntse ho tlosoa litšila tse ling.

Mokhoa oa moloko oa bobeli o nolofatsa ts'ebetso motheong oa ts'ebetso e tsitsitseng, o fokotsa kotsi ea kotsi, 'me o ntlafatsa katleho ea ho ntšoa ha RNA.

Lihlahisoa tsa moemeli: QIAGEN RNeasy le lihlahisoa tse ngata tsa malapeng

Leha ho le joalo, mekhoa ea bobeli ea moloko oa pele le oa bobeli e hloka hore ho sebelisoe DNase ho tlosa tšilafalo ea DNA, e leng se tlisang tšitiso e itseng litekong tse latelang tse tlaase.Masala a DNase a tla fokotsa ts'ebetso ea ho fetolela ka morao le ho fokotsa motsoako oa pele oa cDNA, ka hona e ama ho nepahala ha liphetho.

Ka hona, mokhoa oa ho ntša moloko oa boraro o ntlafalitsoe motheong oa moloko oa bobeli.Teko ha e hloke ho eketsa li-enzyme, 'me RNA e ka fumanoa habonolo ka litšiea tse peli feela.

Kholomo 1: DNA-Cleaning Column ho hlakola DNA

Kholomo 2: Kholomo ea RNA-feela e bapatsa le ho khutlisa RNA

Ts'ebetso e bonolo joalo ha e boloke nako feela, empa e boetse e thusa ts'ebetso ea liteko tse tlase!

Lihlahisoa tsa moemeli: QIAGEN RNeasy Plus leForegeneLihlahisoa tsa letoto la tlhoekiso ea RNA

(Reasy Plus)

（'Mele oa ForegenePlant Total RNA Isolation Kit）

Ke u bolelle ka khutso!Foregene's RNA kit e boetse e hlahisa RNase Eraser e bonolo haholo ho e sebelisa, e ka tlosang 400μg ea RNase ka botlalo sebakeng se tiileng ka motsotso o le mong, ntle le ho kenya lintho tse chefo 'meleng oa motho!

Setšoantšo1: Chate ea papiso ea ts'ebetso ea ts'ebetso

Lihlahisoa tsa Foregene li ka tlatsa tlhahiso ea RNA ka lipina tse peli feela, u se ke ua etsa hore ba bang ba boulele tsoelo-pele ea hau ea liteko!

Setšoantšo2:Eliphetho tsa liteko papiso chate

Moloko oa pele (1: TRIzol)

Moloko oa bobeli (2: RNeasy of QIAGEN)

Moloko oa boraro (3: QIAGEN's RNeasy plus, 4: Foregene)

Setšoantšo3: Kerafo ea qPCR (kerafo e holisang curve)

Moloko oa pele (botala bo bolela TRIzol -, bofubelu bo bolela TRIzol +)

Moloko oa bobeli (boputsoa ba leholimo bo bolela QIAGEN RNeasy-, bofubelu bo bolela QIAGEN RNeasy +)

Moloko oa boraro (boputsoa bo bolela QIAGEN RNeasy plus, botala bo bolela Foregene)

(Tlhokomeliso: qPCR graph “-” e bontša hore DNase ha e e-s’o eketsoe ’me tšilafalo ea DNA ha e so tlosoe. Ka hona, CT e phahame ’me sephetho ha sea nepahala; “+” e bontša hore DNase e kentsoe ’me tšilafalo ea DNA e tlositsoe, sephetho se nepahetse)

Ntlafatso e 'ngoe le e 'ngoe ea tekheniki e bolela hore ts'ebetso ea teko e ntse e tsoela pele ka tsela e bonolo le e bonolo ho basebelisi.

Foregene e ikemiselitse ho thusa bafuputsi ba bangata ba saense ho boloka nako ea liteko le litšenyehelo, ho fumana liphetho tsa liteko tsa boleng bo holimo, 'me ka kopanelo ba thehe likatleho tsa bohlokoa tsa lipatlisiso tsa mahlale.