Recommended Procedures for the Extraction of RNA
Jan PedersenUSDA, 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
Processing Method
Notes
Gallinaceous Poultry (chickens, turkeys, quail)
Tracheal swab Ambion or RNeasy RNA extraction, then RRT-PCR
Virus primarily replicates in the respiratory tract (LPAI)
Waterfowl-ducks
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
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 96specimens with Ambion reagents in a single run (20 min)
Studies have demonstrated equivalency with the manualAmbion procedure-Similar Ct-No evidence of cross contamination
Requires equipment specificconsumables
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)
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