Isingeniso:

I-RNA, igama lesayensi i-ribonucleic acid, isifinyezo sayo sivela ku-RiboNucleic Acid, iwuhlobo lwe-nucleic acid olwakhiwe ngokuxhuma okungenani inqwaba yama-ribonucleotides ngamabhondi e-phosphodiester.

Isisekelo sokuhlola i-RNA ukukhipha Ingqikithi ye-RNA engangcolisi, futhi umgomo oyisisekelo wokukhishwa kwe-RNA ungafinyezwa kanjena:

1. Phakamisa amaseli kuqala

2. Bese ususa ukungcola okuyi-macromolecular (amaprotheni, ama-lipids, i-DNA, njll.)

3. Okokugcina, i-RNA iyana ngenkathi ikhipha usawoti nezincibilikisi eziphilayo, futhi iqhubeka nokuhlanza i-RNA.

Indlela yokukhipha eyedlule neyamanje

Isizukulwane sokuqala sendlela yokukhipha i-RNA yindlela ye-TRIzol yemvula (ama-reagents asetshenziswayo ahlanganisa i-guanidine isothiocyanate, i-phenol, i-chloroform, njll.) eyasungulwa umkhulu okuthiwa u-Piotr Chomczynski nogogo u-Nicoletta Sacchi, ngoba lokhu okusungulwe kuvumela ukukhipha kwe-RNA Ngokushesha futhi kulula, kusukela ngaleso sikhathi kwaqala ukucwaninga kwe-RNA.

Kamuva, uBabamkhulu wathengisa leli lungelo lobunikazi ku-Invitrogen, futhi i-Invitrogen yathuthukisa le ndlela emhlabeni futhi yaba indlela esetshenziswa kakhulu.

Kamuva, i-Invitrogen yatholwa yi-Thermo!

Umkhiqizo omele: I-Invitrogen's TRIzol reagent

Kodwa-ke, indlela ye-TRIzol yokusana idinga imvula eminingi kanye nokukhipha i-RNA, futhi ama-reagents asetshenzisiwe anobuthi futhi athatha isikhathi eside ukusebenza.

Ngakho-ke, indlela yokuhlanza ikholomu ye-enzyme yesizukulwane sesibili yaba khona, kusetshenziswa ulwelwesi lwejeli ye-silica njengento ethile ye-nucleic acid adsorption ukuze kukhuliswe ukutholwa kwe-RNA kusampula ngenkathi kususwa okunye ukungcola.

Indlela yesizukulwane sesibili yenza umsebenzi ube lula ngesisekelo sokusebenza okuzinzile, inciphisa isici sobungozi, futhi ithuthukise ukusebenza kahle kokukhipha i-RNA.

Imikhiqizo emele: QIAGEN RNeasy kanye nemikhiqizo eminingi yasekhaya

Kodwa-ke, zombili izindlela zesizukulwane sokuqala nesesibili zidinga ukusetshenziswa kwe-DNase ukuze kususwe ukungcoliswa kwe-DNA, okuletha ukuphazamiseka okuthile ekuhloleni okulandelayo komfula.I-DNase eyinsalela izonciphisa ukusebenza kahle kokuhlehla kokuloba futhi inciphise ukuhlanganiswa kwe-cDNA yomucu wokuqala, ngaleyo ndlela kuthinte ukunemba kwemiphumela.

Ngakho-ke, indlela yokukhipha isizukulwane sesithathu ilungiselelwe ngesisekelo sesizukulwane sesibili.Ukuhlolwa akudingi ukwengeza noma yimaphi ama-enzyme, futhi i-RNA ingatholakala kalula ngamakholomu amabili kuphela.

Ikholomu 1: Ikholomu Yokuhlanza I-DNA ukuze kusule i-DNA

Ikholomu 2: Ikholomu ye-RNA kuphela ikhangisa futhi ibuyisele i-RNA

Ukusebenza okulula okunjalo akugcini nje konga isikhathi, kodwa futhi kusiza ukusebenza kwezivivinyo ezingaphansi komfula!

Imikhiqizo emele: QIAGEN RNeasy Plus kanyeI-ForegeneImikhiqizo yochungechunge lokuhlanza i-RNA

(I-RNeasy Plus)

(I-Foregene'sIkhithi Yokuhlukanisa Yezitshalo Yesamba se-RNA)

Ngikutshele buthule!Ikhithi ye-RNA ye-Foregene futhi iveza Isisuli se-RNase okulula kakhulu ukusisebenzisa, esingasusa ngokuphelele u-400μg we-RNase endaweni eqinile ngomzuzu ongu-1, ngaphandle kokwengeza noma yiziphi izinto ezinobuthi emzimbeni womuntu!

Isithombe1: Ishadi lokuqhathanisa lenqubo yokusebenza

Imikhiqizo ye-Foregene ingaqedela ukukhipha kwe-RNA ngezingoma ezimbili kuphela, ungenzi abanye babe nomona ngenqubekelaphambili yakho yokuhlolwa!

Isithombe2:Eimiphumela ye-xperimental ishadi lokuqhathanisa

Isizukulwane sokuqala (1: TRIzol)

Isizukulwane sesibili (2: RNeasy of QIAGEN)

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

Isithombe3: igrafu ye-qPCR (igrafu yejika yokukhulisa)

Isizukulwane sokuqala (okuluhlaza kusho i-TRIzol -, okubomvu kusho i-TRIzol +)

Isizukulwane sesibili (isibhakabhaka esiluhlaza sisho QIAGEN RNeasy-, okubomvu kusho QIAGEN RNeasy +)

Isizukulwane sesithathu (okuluhlaza okwesibhakabhaka kusho QIAGEN RNeasy plus, okuluhlaza kusho iForegene)

(Qaphela: igrafu ye-qPCR ethi “-” ibonisa ukuthi i-DNase ayizange yengezwe futhi ukungcoliswa kwe-DNA akukasuswa. Ngakho-ke, i-CT iphakeme futhi umphumela awunembile; “+” ubonisa ukuthi i-DNase yengeziwe futhi ukungcola kwe-DNA kususiwe, umphumela unembile)

Konke ukuthuthukiswa kobuchwepheshe kusho ukuthi ukusebenza kokuhlolwa kuthuthukiswa ngendlela elula nelula kakhulu umsebenzisi.

I-Foregene ihlose ukusiza abacwaningi besayensi abengeziwe bonge isikhathi sokuhlola nezindleko, bathole imiphumela yokuhlola yekhwalithi ephezulu, futhi ngokuhlanganyela badale impumelelo yocwaningo lwesayensi olubaluleke kakhulu.