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iindaba716 (1)

Ngaba usasebenzisa i-Trizol ukukhupha i-RNA?
Gosh, ungandivumeli ndikhathazeke ngempilo yakho!
UTrizol usezobekwa ecaleni, ungandixeleli ukuba usebumnyameni?

Icandelo eliphambili le-Trizol reagent yi-phenol, kunye ne-organic reagents ezifana ne-chloroform nazo zongezwa kumsebenzi wokulinga.Ukuvezwa kwexesha elide kuya kubangela ingozi kwimpilo yakho, ngakumbi ukudibana kwesikhumba ngokuthe ngqo kunokubangela ukutshisa okukhulu kweekhemikhali kunye nezibonda ezingapheliyo.

Phantsi kweemeko eziqhelekileyo, amaqabane aselebhu anokunciphisa kuphela iingozi ngokuzixhobisa ngokupheleleyo (ukunxiba iidyasi zaselebhu, iiglavu kunye nemaski).

Kwaye ingcebiso yam kuwe yile: nceda ubuyisele i-Trizol ngokuthe ngqo ukunqanda ingozi evela kumthombo.

1. Imveliso yesizukulwana sokuqala sokutsalwa kwe-RNA
Phuma ngo-1987 (ii-reagents ze-Trizol)

inzuzo:
Ukutsalwa okupheleleyo ngakumbi

Izinto ezingeloncedo:
Ii-reagents ezinobuthi ezifana ne-guanidine isothiocyanate-phenol-chloroform ziyafuneka;

Ukusebenza nzima;
Kufuneka usebenzise i-DNase ukususa ungcoliseko lwe-DNA

2.Imveliso yesizukulwana sesibini sokutsalwa kwe-RNA
Phuma ngo-1994 (QIAGEN RNeasy kunye neemveliso ezininzi zasekhaya)

inzuzo:
Indlela yekholomu ye-centrifugal yamkelwa, ekulula ukuyisebenzisa;

Ukusilela:
Kufuneka i-centrifuge ukususa inkunkuma yezicubu

Kufuneka usebenzise i-DNase ukususa ungcoliseko lwe-DNA

3.Imveliso yesizukulwana sesithathu sokutsalwa kwe-RNA
Iphuma kwi-2005 (QIAGEN RNeasy Plus kunye noForegeneIimveliso zokuhlanjululwa kwe-RNA)

inzuzo:
Sebenzisa ngokuthe ngqo ikholamu yokususa i-DNA ukususa i-DNA kunye ne-tissue debris ngexesha elifanayo, ukugcina ixesha kunye nomgudu;

Ikhuselekile kwaye ifanelekile, akukho mfuneko yokongeza ikholamu yokucoca kunye ne-DNase ukususa ngokulula ukungcola kunye nokuphepha ukungcoliseka kweDNA.

iindaba716 (2)

Makhe sijonge umahluko wokusebenza phakathi kwemveliso yesizukulwana sokuqala (i-Trizol), imveliso yesizukulwana sesibini (i-RNeasy) kunye nemveliso yesizukulwana sesithathu (i-Forgene) ye-RNA extraction:iindaba716 (3)

Ukusebenza kumzobo kucacile: imveliso yesizukulwana sesithathu ineenzuzo ezicacileyo

Amagama amathathu nje ukuchaza: kulula!Khawuleza!Ukhuseleko

Itshathi ye-PK yeziphumo zovavanyongenxaisizukulwana sokuqala, isizukulwana sesibini kunye nesesithathu isizukulwana semveliso yeRNA:

1. Electrophoretogram

Isizukulwana sokuqala (1: Trizol)
Isizukulwana sesibini (2:QIAGEN RNeasy)
Isizukulwana sesithathu (3: QIAGEN RNeasy plus, 4: Foregene)

iindaba716 (4)

Luluphi uhlobo lwemveliso olulungileyo, uya kukwazi ngokuthelekisa
I-RNA ikhutshwe kusetyenziswa ikhithi ye-Foregene
Akunjalo kuphela ukugxila okuphezulu kodwa kunye nokunyaniseka okulungileyo

Igrafu ye-2.qPCR (igrafu yegophe yokukhulisa)

iindaba716 (5)
Isizukulwana sokuqala (iTrizol)

iindaba716 (6)

Isizukulwana sesibini (isibhakabhaka esiluhlazakuthethaQIAGEN RNeasy-, ebomvukuthethaQIAGEN RNeasy +)

iindaba716 (7)

Isizukulwana sesithathu (ibhlowu ithetha iForegene, eluhlaza ithetha QIAGEN RNeasy plus)

(Qaphela: Isizukulwana sesibini se-QIAGEN RNeasy yegrafu ye-qPCR ithetha ukuba ikiti ayifaki i-DNase kwaye ayisusi ukungcoliswa kwe-DNA. Ngoko ke, i-CT iphezulu kwaye umphumo awuchanekanga; I-QIAGEN RNeasy + ithetha ukuba i-DNase yongezwe kwaye ukungcola kwe-DNA kususiwe, kwaye umphumo uchanekile)

Nokuba yi-electrophoretogram okanye igrafu ye-PCR yokukhulisa i-curve, umgangatho weemveliso ze-Foregene awukho ngaphantsi kwiimveliso zangaphandle.


Ixesha lokuposa: Jul-16-2021