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)