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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.