Recommended Procedures for
the Extraction of RNA
Jan Pedersen
USDA, APHIS, VS,
National Veterinary Services Laboratories,
Ames, IA 50010
RNA Extraction



Isolates RNA from other cellular components
in the sample
Removes inhibitory substances – may not
eliminate all
Inactivates endogenous RNases in specimen
(guanidine isothiocyanate in lysis solution)
 Qiagen® silica column
 Ambion® magnetic bead
 Trizol® – monophasic organic solution
Extraction
Obtaining high quality RNA is the 1st and most important step

Proper handling and use of RNase-free materials will eliminates
degradation of RNA and introduction of RNases

RNases are ubiquous enzymes which degrade RNA

Storage of isolated RNA
 Store in RNase – free solution
 24 hr. - Store at 4 C
 >24 hr. - Store at -70 C
RNase Contamination





Body fluids such as
perspiration
Pipette tips and
tubes
Lab surfaces and
environment
Water and buffer
Endogenous RNA





Powder free gloves
Use certified RNase –
free tips and tubes
Dust, bacteria,
spores, etc.
Use RNase-free water
Proper handling &
storage of specimens
Specimen Processing &
RNA Extraction
Lysis step inactivates virus






Ambion
1st step in BSC
Qiagen – lysis reagent
contains BME
Vented BSC
Trizol -contains phenol
Vented BSC
Sample types and processing methods
Species/
Sample Type
Preferred
Specimen
Gallinaceous
Tracheal swab
Poultry (chickens,
turkeys, quail)
Processing
Method
Notes
Ambion or RNeasy
RNA extraction,
then RRT-PCR
Virus primarily replicates in the
respiratory tract (LPAI)
Cloacal Swab
Ambion or Trizol
Reagent RNA
extraction, then
RRT-PCR
Virus primarily replicates in the
intestinal tract. RNA extraction
method must be modified for
cloacal samples
Any species
Tissue samples
Trizol Reagent RNA
extraction, then
RRT-PCR
For HPAI viruses high levels of
virus may be in tissues.
Environmental
samples
(Swab)
Virus isolation,
RRT-PCR not
recommended
RRT-PCR can detect inactivated
virus
Waterfowlducks
Swabs and Species





Swab type and size - avoid calcium alginate and swabs
with wooden shafts (may contain PCR inhibitors)
Small birds make it difficult to collect enough material
for efficient extraction
Cloacal samples typically recommended for waterfowl
TR/OP swabs are acceptable for the surveillance of
Asian H5N1 in waterfowl and wild birds
RRT-PCR not recommended for environmental
swabbing when you want to assure facility is free of
infectious virus
Magnetic Bead RNA Extraction




Paramagnetic beads with nucleic acid binding surface
are used to bind RNA following lysis
Bead with RNA is captured on magnets and the
supernatant containing cell debris and other
contaminants is removed with washes
High throughput – 96 well format
Equivalent sensitivity to Qiagen procedure for TR
swab specimens, but is a more sensitive procedure
for CL swab specimens
Magnetic Bead RNA Extraction (1)



Add 101µl
lysis/binding solution
to specimen well.
Add 50µl swab
specimen to the
lysis/binding solution
Shake for 30 sec.
Lysis step will rupture the cell membrane and release
cellular components and nucleic acid from the cell
Magnetic Bead RNA Extraction (2)


Add 20µl
resuspended
magnetic beads to
each well
Shake for 4 min.
Magnetic Bead RNA Extraction (3)





Capture RNA binding
beads on magnetic
stand for 2 min.
Discard supernatant
Remove plate from
magnetic stand
Add 100µl wash
solution 1
Shake for 30 sec.
Magnetic Bead RNA Extraction (4)




Pellet the beads for
1 min. and remove
wash supernatant
Add 100µl wash
solution II
Shake for 30 sec.
Repeat wash II
procedure
Magnetic Bead RNA Extraction (5)




Following the 2nd wash
II step dry the beads by
shaking vigorously for 2
min.
All residual ETOH must
be removed in dry step
Add 50µl elution buffer
and shake for 4 min.
Collect the beads on the
magnetic stand and
transfer RNA to tube.
KingFisher Magnetic Particle
Processor
Will extract RNA from 24 or 96
specimens with Ambion
reagents in a single run (20 min)
Studies have demonstrated
equivalency with the manual
Ambion procedure
-Similar Ct
-No evidence of cross
contamination
Requires equipment specific
consumables
Qiagen Silica Column RNA Extraction
•Conducted in BSC II hood with 500µl of diagnostic sample
•Reagent and wash solutions are processed through column with vacuum
manifold or with centrifugation
•Efficient for TR/OP swabs, however CL swabs can be problematic
Add 500 µl RLT lysis buffer to specimen and vortex well
Add 500 µl 70% ETOH to lysed specimen and vortex
Centrifuge at 5000 x g for 5 min.
Following lysis, addition of ETOH & centrifugation the specimen is added
to column
*Open lid of column before turning pump on – to prevent damage to silica
membrane
Apply entire content of specimen to column – do not disturb the pellet
Wash RNA with buffer – 700 µl RW1 (1X) followed by 500 µl RPE (2X)
Turn vacuum pump off
Remove column and spin to remove ETOH and dry membrane
Important - Residual ETOH is inhibitory to PCR
Add 50 µl of RNase-free H2O to silica membrane
Do not touch membrane with tip, changing pipette tips between each
column
Incubate 1-5 min. and centrifuge to elute RNA
TRIZOL



Ready to use reagent for the isolation of
total RNA
Mono-phasic solution of phenol and
guanidine isothiocyanate
An improvement on the single-step RNA
isolation method developed by
Chomczynski & Sacchi (Anal. Biochem,
162. 1987)
Trizol Extraction Cont.




Chloroform is added for phase separation
allowing collection of the aqueous phase
containing RNA
RNA is precipitated with addition of Isopropyl
RNA precipitate is often invisible before
centrifugation but may forms a gel-like pellet
on the side and bottom of the tube
Final wash with ethanol
RNA Pellet




Briefly dry pellet for 5-10 min.
Do not let pellet dry completely or over-dry as
this will decrease solubility however, all
residual ETOH must be removed
Reconstitute pellet with RNase-free water and
incubate at least 1 hr.
Vortex reconstitute pellet prior to pipetting
Wet Lab Experience



Each person will extract the RNA from
two specimens (1, 2, or 3) using the
Ambion magnetic bead procedure
The Qiagen silica column procedure will
be demonstrated
Trizol is described in detail in protocol
Magnetic Bead RNA Extraction







Add magnetic beads which are fully
suspended in binding solution
Capture beads on magnetic stand
Remove supernatant
Perform 2 wash steps with ethanol
Dry beads to remove ethanol
Remove nucleic acid from paramagnetic
beads with elution buffer
Pellet beads and remove RNA (50µl)