RNA isolation from whole blood

There are two steps in synthesis of cDNA:1. Total RNA is to be isolated from the white blood cells present in the blood using Oligotex
Direct mRNA mini kit of Genetix (MBI Fermentas) (P-3) Price Rs.22445/=, Cat No.72022
(2005-06) for 12 reaction or by using Trizol method. Trizol method works very well as it is
simple and less expensive (Used by Dr. Sahoo).
2. Reverse transcription-polymerase chain reaction (RT-PCR) to be carried out for cDNA
synthesis using specific primers and total RNA by following the kit protocol. (Quigen One
Step RT-PCR Kit-25 reactions, Price Rs.12230/=, P-4, Cat No.210210 of Genetix-MBI
Fermentas).
Different steps followed in the protocol are given below:
1.
1.

Separation of White blood cells:

Blood sample was collected in fresh15 ml poly propylene tube with anticoagulant
(2.7% EDTA, 500 l for 5 ml blood) and immediately perform the next step (RNA
will degrade very fast, so to get more yield, quickly go to next step). Bring to lab on
ice pack, keep at 4oC for an hour if needed and perform next step within this time.

2.

To make volume add autoclaved PBS (Phosphate Buffer Saline, pH 7.5) to blood and
mix well, alternatively blood samples can be diluted 1:1 with PBS.

3.

Add 3 ml histopaque 1077 density gradient solution (Sigma) to fresh 15 ml tube if

working in 50 ml tube add 15 ml histopaque so that the level of histopaque reaches
above the triangular base of the tube.

4.

IMP- Layer the blood on histopaque only when it comes to room temperature (35 oC37oC) it is not used chilled or freezed. Dont keep histopaque tubes on ice.

5.

From the sides of the tube slightly and slowly pour the blood over the histopaque so
that the blood forms a layer over histopaque. The two layers should be visible
separately one as transparent histopaque and the blood above. If histopaque get mixed
do not proceed further and discard the sample. Always pour blood samples after
proper mixing it so that cells get into suspension.

6.

Centrifuge the tubes at 2000-2300 rpm range for 30 min on room temperature.

7.

After centrifugation three layers are seen above is plasma either transparent or with
yellow or red tinge, beneath it a cloudy layer of 1-1.5 cm thickness of WBC and
below will be RBC.

8.

The cloudy buffy coat/ layer (white ring of WBC was appeared at the interphase of
plasma (upper side ) and RBCs& histopaque (bottom) ) was taken in another tube
(First plasma was discarded using pipette and the WBC were taken out by using same
tip used for plasma)

9.

In the separated WBC of step 5, 12ml of PBS was added (Same used for washing of
cells (Dr. Ajit)). And mixed gently by tilting the tube.

10.

The tube was centrifuged at 1800rpm for 15 min at room temperature.

11.

The supernatant was discarded and the WBC pellet was collected.

2.
1.

Isolation of Total RNA using TRIzol reagent:

The complete WBC pellet obtained in previous step was taken in a DEPC (0.1%) /
Chloroform (10 %) RNAs inhibitor treated 1.5 ml micro centrifuge tube after proper
mixing the WBC with residual PBS by repeated mixing with 1 ml pipette and 1 ml of
RNA-Xpress reagent (Himedia, Cat no.MB601-100ML,) was added.

2.

The content was mixed by tilting the tube and then it was homogenized by 1 ml pipette
tip.

3.

The homogenate was then mixed thoroughly by vortexing and incubated for 5 min at 15
to 30oC (or keep aove ice pack or ice or in 4 oC in fridge) to permit dissociation of
nucleoprotein complexes.

4.

To the homogenate 5l of 20% Proteinease K (Proteinase-K, 20 mg) was added.

5.

PAUSE after adding trizol and proteinase K the RNA becomes stable, hence the
method can be paused at this time, The mixture of WBC + Trizol + Proteinase K can be
kept at -21 oC for 1-2 hours without any deterioration of the sample and incubation of 1
hr also increase RNA yield)

6.

The homogenate was then thawed on ice (If frozen previously) and add 0.2ml of
chloroform to it, mix be inversion and brief vortexing.

7.

Incubated at room temperature (or at 4 oC or over ice or ice pack; RT here means 30 oC,
higher temperatures in summer may cause deterioration) for 10 minute

8.

Samples were centrifuged @12000 rpm for 15 minutes at 4 oC (cooling microfuge).

9.

If centrifuge machine does not have pre cool function, run it empty without samples for
2 min at 4oC at 12000 rpm so that the chamber temperature becomes cool.

10.

Following centrifugation; the mixture separates into a lowers: red chloroform phase, a
white interphase and a colourless upper aqueous phase. RNA remains exclusively in the
upper aqueous phase.

11.

The upper aqueous phase was transferred to a fresh tube and adding 0.5ml of 100%
isopropyl alcohol, and kept for 10 min at 4 oC or if required, it can be kept for one hour
at -21oC.

12.

Then the sample were centrifuged at 12000 rpm for 12 minutes at 4 oC. The white
pellet will form in the bottom. May not be visible sometime or may not be exactly white
it may be cloudy or very faint.

13.

The supernatant was discarded and 70% ethanol was added on the top of the pellet, kept
in freezer for 2 minutes to remove salts and centrifuged at 12000 rpm for 10 minutes at
4 oC.

14.

Finally after discarding the supernatant, the pellet was dried, do not desiccate the pellet,
if pellet is visible from eye pipette out the ethanol carefully by 10 ul pipette, or if it is
not visible discard ethanol by tilting and then keep on ice or at 30 35oC until ethanol is
not visible, over drying may kill RNA. Keep the DEPC treated autoclaved nuclease free
water in water bath at 60oC so that it gets heat up until the RNA pellet dries out. Once it
gets dry, add 20-25 ul of DEPC treated water on the pellet mix by pipette itself and keep
on ice immediately.

3.

Rapid agarose gel electrophoresis for RNA quality

RNA can be rapidly checked on agarose gel if u doesnt have access to spectrophotometer.
1. Keep aliquot 3-4 ul of RNA in PCR tube from the stock after dissolving the pellet and keep
rest of the sample at -21oC along with aliquotted samples. Samples are aliquotted to avoid
repeated thawing of stock RNA.
2. Gel assemble should be clean with chloroform alongwith combs and gel tray.
3. Run this gel on 1X TAE buffer it is better for RNA runs.
4. The 1X TAE buffer used to dissolve agarose should be made in DEPC treated (0.1%
DEPC) autoclaved distil water, rest buffer to fill up tank can be made in simple autoclaved
distil water.
5. Make 1-1.5% agarose gel same as done in DNA runs.

6. When everything is ready take out your RNA sample from freezer keep on ice/ ice pack/
mini cooler add 1 ul loading dye to each sample tube mix and load in the gel with 100bp
ladder, run at 80- 100 V for 45 min or hour. USE PIPETTE & TIPS THAT ARE DEPC
TREATED CAREFULLY NOT TO MIX THEM WITH DNA WORKING TIPS AND
PIPETTE, AFTER USE IMMEDIATELY CLEAN PIPETTE WITH CHLOROFORM AND
KEEP BACK IN RNA WORKING AREA.
7. See the gel under gel doc and take the image.

Residual DNA

16s rRNA
m RNA
24s rRNA

100 bp ladder

Good quality RNA shows two intact bands (16s and 24s rRNA) with smearing in
between (mRNA).