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 columnAmbion® magnetic beadTrizol® – monophasic organic solution

ExtractionObtaining 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 RNAStore in RNase – free solution24 hr. - Store at 4 C >24 hr. - Store at -70 C

RNase Contamination

Body fluids such as perspirationPipette tips and tubesLab surfaces and environmentWater and bufferEndogenous RNA

Powder free gloves

Use certified RNase –free tips and tubesDust, bacteria, spores, etc.Use RNase-free waterProper handling & storage of specimens

Specimen Processing &RNA Extraction

Lysis step inactivates virusAmbion1st step in BSC

Qiagen – lysis reagent contains BME Vented BSC

Trizol -contains phenolVented BSC

Sample types and processing methods

RRT-PCR can detect inactivated virus

Virus isolation, RRT-PCR not recommended

(Swab)Environmental samples

For HPAI viruses high levels of virus may be in tissues.

Trizol Reagent RNA extraction, then RRT-PCR

Tissue samplesAny species

Virus primarily replicates in the intestinal tract. RNA extraction method must be modified for cloacal samples

Ambion or Trizol Reagent RNA extraction, then RRT-PCR

Cloacal SwabWaterfowl-ducks

Virus primarily replicates in the respiratory tract (LPAI)

Ambion or RNeasy RNA extraction, then RRT-PCR

Tracheal swabGallinaceous Poultry (chickens, turkeys, quail)

NotesProcessing Method

Preferred Specimen

Species/ Sample Type

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 extractionCloacal samples typically recommended for waterfowlTR/OP swabs are acceptable for the surveillance of Asian H5N1 in waterfowl and wild birdsRRT-PCR not recommended for environmental swabbing when you want to assure facility is free of infectious virus

Magnetic Bead RNA ExtractionParamagnetic 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 solutionShake for 30 sec.

Lysis step will rupture the cell membrane and release cellular components and nucleic acid from the cell

Live virus in specimen is inactivated and remainder of procedure can be conducted outside of BSC

Magnetic Bead RNA Extraction (2)

Add 20µl resuspendedmagnetic beads to each well

Shake for 4 min.

• Combine sample with lysis solution• Shake• Add beads/binding mixture• Capture beads and remove supernatant• Conduct 2 wash steps• Shake to remove ethanol• Add elution buffer

Magnetic Bead RNA Extraction (3)

Capture RNA binding beads on magnetic stand for 2 min.Discard supernatantRemove plate from magnetic standAdd 100µl wash solution 1Shake for 30 sec.

Magnetic Bead RNA Extraction (4)

Pellet the beads for 1 min. and remove wash supernatantAdd 100µl wash solution IIShake 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 stepAdd 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 RNAMono-phasic solution of phenol and guanidine isothiocyanateAn improvement on the single-step RNA isolation method developed by Chomczynski & Sacchi (Anal. Biochem, 162. 1987)

Sample Lysis With Trizol Reagent

Sample: homogenized tissue

Cell Lysis

Disruption of cells and Dissolving of cell components

Trizol Extraction Cont.

Chloroform is added for phase separation allowing collection of the aqueous phase containing RNARNA 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 tubeFinal 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 removedReconstitute 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 procedureThe Qiagen silica column procedure will be demonstratedTrizol is described in detail in protocol

Magnetic Bead RNA Extraction

Add magnetic beads which are fullysuspended in binding solutionCapture beads on magnetic standRemove supernatantPerform 2 wash steps with ethanolDry beads to remove ethanolRemove nucleic acid from paramagnetic beads with elution bufferPellet beads and remove RNA (50µl)

Qiagen Silica Column Extraction

Add 500µl RLT lysis buffer to 500µl specimen in tubeVortex for 15 sec.Pulse spin to remove liquid from lidAdd 500µl 70% ETOHVortex wellCentrifuge at 5,000 x g for 5 min.