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 1 st 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 1 st 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 swabAmbion or RNeasy RNA extraction, then RRT-PCR Virus primarily replicates in the respiratory tract (LPAI) Waterfowl- ducks Cloacal SwabAmbion 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 speciesTissue samplesTrizol 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

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

Magnetic Bead RNA Extraction (2)  Add 20µl resuspended magnetic 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 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 2 nd 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 H 2 O 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, )

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 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)

Qiagen Silica Column Extraction  Add 500µl RLT lysis buffer to 500µl specimen in tube  Vortex for 15 sec.  Pulse spin to remove liquid from lid  Add 500µl 70% ETOH  Vortex well  Centrifuge at 5,000 x g for 5 min.