19 Isolation of total RNA:TRIzolTM and a secondary
purification using the RNAeasyTM columns UREAD
SopID: 19 - UREAD
NuGO approved 2008
Transcriptomics
Keywords: RNA isolation, TRIzol, RNeasy
When citing this SOP you should acknowledge both NuGO and the appropriate NuGO
partner institution that has made the SOP available. Please use a form of words such as:
We used the NuGO Standard Operating Procedure (SOP) number 19 produced by the
University of Reading Details of the SOP are available via the web link:
http://www.nugo.org/frames.asp?actionID=28250&action=loginFromPP
For more details please contact University of Reading
Backgrounds
Extraction of good quality RNA is vital to the production of of high quality expression data. We suggest
that you take all the usual precautions during the preparation of the RNA to prevent degradation or
contamination.
Overview
This method guarantees a high purity of RNA. The first step uses TRIzolTM and a secondary
purification using the RNAeasyTM columns. This procedure shows a significant improvement in purity
of cDNA, and it is believed that the final column purification step may remove residual components
from the RNA that may inhibit one or more of the enzymes used in cDNA synthesis reactions. In
addition, this procedure has the advantage that it combines the ability of the TRIzol reagent to
instantly stop any transcriptional change that might occur during the harvest of the material for RNA
extraction, with the ability of the spin columns to remove genomic DNA and all contaminants efficiently
and without the need for of the DNAse treatment. The disadvantages are the high cost of the kit and
the reagents, and the lower yield of total RNA, which may be a problem if the starting material is not
very abundant.
Materials

TRIzol reagent (Invitrogen)

Chloroform (Sigma)

Isopropanol (Sigma)

1.5 ml eppendorf tubes

Aerosol-free tips (ART)
Main Procedures
TRIZOL PROTOCOL

Homogenization

Phase Separation

RNA precipitation

RNA Wash

Redissolving the RNA
RNeasy PROTOCOL

Add RLT buffer, then ethanol to the RNA sample

Apply sample to an RNeasy minicolumn and centrifuge

Pipette RPE buffer onto the RNeasy column and centrifuge

Add RPE buffer and centrifuge

Pipette RNase free water onto the RNeasy membrane and centrifuge

Store at -80ºC.
Sub Procedures
TRIZOL PROTOCOL
Homogenization
1)
Add 1ml of TRIzol to 100mg of tissue.
2)
Homogenise in a homogeniser or similar device at half speed for 2 minutes. (For tough tissue
increase the speed and time: CARE)
Phase Separation
3)
Incubate at room temperature for 5 minutes.
4)
Add 0.2 ml chloroform; shake tube vigorously by hand for 15 seconds.
5)
Incubate at 15 - 30ºC for 2 - 3 minutes.
6)
Centrifuge samples at 12000 x g for 15 minutes at 2 - 8ºC.
7)
Transfer aqueous (upper) phase to a new tube (take only 500ul). [The upper phase is 60%
of the volume of TRIzol used for homogenization.]
RNA precipitation
8)
Add 500ul of isopropyl alcohol.
9)
Incubate for 10 minutes at 2 - 8ºC.
10) Centrifuge at high speed for 10 minutes at 2 - 8ºC. The RNA precipitate forms a gel like
pellet on the side/bottom of the tube.
RNA Wash
11) Remove the supernatant and wash the pellet with 1ml of 75% ethanol.
12) Vortex the sample and then centrifuge for 5 minutes at 2 - 8ºC (>10000 g)
13) Remove the supernatant and air dry the RNA pellet for 5 - 10 minutes.
Redissolving the RNA
14) Resuspend in RNase free water. Adjust sample to 100ul volume.
RNeasy PROTOCOL
1)
Add 350 ul RLT buffer (with ß-mercaptoethanol) to the 100ul RNA sample, mix thoroughly.
2)
Add 250ul of 100% ethanol and mix well by pipetting 10 times.
3)
Apply the sample (700ul) to an RNeasy minicolumn placed in a 2ml collection tube.
4)
Close the tube gently and centrifuge for 15 seconds at >= 8000 x g
5)
Discard the flow through and collection tube. Transfer the RNeasy column to a fresh 2ml
collection tube.
6)
Ensure that ethanol has been added to the RPE buffer. Pipette 500ul of RPE buffer onto the
RNeasy column.
7)
Close tube gently and centrifuge for 15 seconds at >= 8000 x g.
8)
Discard the flow through and replace the RNeasy column in the same collection tube.
9)
Add another 500ul of RPE buffer to the RNeasy column.
10) Close the tube gently and centrifuge for 2 minutes at >= 8000 x g to dry the RNeasy
membrane
11) Transfer the RNeasy column into a new 1.5ml collection tube. Centrifuge for 1 minute at full
speed in a microcentrifuge.
12) To elute, transfer the RNeasy column to a fresh 1.5ml collection tube. Pipette 50ul of RNase
free water onto the RNeasy membrane.
13) Close the tube gently and centrifuge for 1 minute at >=8000 x g.
14) Add a second 50ul volume of RNase free water to the column and centrifuge for a further
minute at >=8000 x g.
15) The eluted RNA sample is ready for storage. At this stage RNAse inhibitor SUPERase-IN
(Ambion #2694) can be added if required. Add 1ul SUPERase-IN per 30ul of RNA. Store at 80ºC.
Safety
Users must comply with COSHH and local safety regulations.
RNeasy protocol – Carry out the addition of ß-mercaptoethanol to the RLT buffer in a fume hood and
wear appropriate protective clothing.
Additional Information
RNeasy protocol
NOTE: If mRNA is to be isolated it may not be necessary to perform this additional step and instead
use an mRNA isolation kit.
Before proceeding with this protocol it is necessary to add ß-mercaptoethanol to the RLT buffer. In a
fume hood with appropriate protective clothing add 10ul of ß-mercaptoethanol per 1ml of RLT buffer.
This buffer is stable for one month after preparation. Ensure ethanol has been added to the RPE
buffer before use. Add 4 volumes of 100% ethanol (as indicated on the bottle) before use.