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RNA Isolation Using TRIzol Method

This document describes the process of isolating RNA using the TRIzol reagent. TRIzol works by maintaining RNA integrity during tissue homogenization while disrupting cells. The homogenized sample is mixed with chloroform, separating into aqueous and organic phases. RNA remains in the aqueous phase. RNA is then precipitated from the aqueous phase using isopropyl alcohol and centrifugation. The RNA pellet is washed with ethanol and dissolved in RNase-free water. The purpose is to isolate high quality RNA for downstream molecular biology experiments such as detecting gene expression of viruses like hepatitis C and HIV.

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455 views12 pages

RNA Isolation Using TRIzol Method

This document describes the process of isolating RNA using the TRIzol reagent. TRIzol works by maintaining RNA integrity during tissue homogenization while disrupting cells. The homogenized sample is mixed with chloroform, separating into aqueous and organic phases. RNA remains in the aqueous phase. RNA is then precipitated from the aqueous phase using isopropyl alcohol and centrifugation. The RNA pellet is washed with ethanol and dissolved in RNase-free water. The purpose is to isolate high quality RNA for downstream molecular biology experiments such as detecting gene expression of viruses like hepatitis C and HIV.

Uploaded by

zenab saqib
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd

Isolation of RNA

AHLAM NAVEED
17461507-002
Principle

 The isolation of RNA with high quality is a crucial


step required to perform various crucial steps
required to perform various molecular biology
experiment.
 TRIzol reagent is a ready-to-use reagent used for
RNA isolation from cells and tissues.
 TRIzol works by maintaining RNA integrity during
tissue homogenization, while at the same time
disrupting and breaking down cells and cells
components.
Continue ………

 RNA is in the aqueous phase.


 After transferring the aqueous phase, RNA can be
recovered by precipitation with isopropyl alcohol.
Materials required

Reagents :-
 Chloform without any additive.
 Isopropyl alcohol.
 75% ethanol.
 Rnase free water or 0.5% SDS solution.
Procedure

 Homogenization:-
Growth Medium on the cells was
discarded and cells were washed with ice cold1X PBS.
The monolayer was then covered with 1ml of 1 TRIzol
and the cells were lysed and homogenized by repeated
pipetting.
2: Phase Separation

 The homogenized samples were incubated for


5minutes at 15 to 30 C for the complete dissociation
of nucleoprotein complexes.
 0.2 ml of chloroform per 0.75ml of TRIzol reagent
was added. The tubes were shaked vigorously by
hand for 15 seconds incubated them for 15 to 30◦C
for 2 minutes.
 The sample were centrifuged for 15 minutes at
12000g (4◦C).
CONTINUE…..

 The aqueous phase was transferred to the other


tubes.
 RNA remains only in the aqueous phase .
 The volume of aqueous phase is about 70% of the
volume of TRIzol reagent used for homogenization.
RNA isolation…
3: RNA precipitation

 T he RNA was precipitated from the aqueous phase


by mixing with 3 microlitre of glycogen and 500 ml
of isopropyl alcohol.
 The mixture was centrifuged for 30minutes 2 to 8◦C .
 The RNA precipitate forms a gel-like pellet on the
side of the tube at bottom.
4: RNA wash

 The supernatant was removed. The RNA pellet was

washedonce with 75% ethanol, adding 900ml of 75%


ethanol per 0.75ml of TRIzol LS reagent used for the
initial homogenization.
 The sample were inverted and mixed and centrifuged
at 12000 rpm for 30 minutes at a 4 degree.
5: Redissolving RNA

 The RNA pellet was dried.

 RNA was dissolved in RNase-free water by passing

the solution through the pipette tip for a few times,


and incubating for 10minutes at 55 to 60◦C.
Purpose
 To detect the gene expression .
 Hepatitis C
 HIV

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