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Protocol
Boom-silica RNA extraction
Keywords: Boom-silica, RNA, extraction, GuSCN, Phenol, Silica
This protocol describes extraction of nucleic acids from serum or organ material for subsequent RT-PCR analysis.
This protocol describes extraction of nucleic acids from serum or organ material for subsequent RT-PCR analysis. The content of PCR-interfering agents is reduced thus resulting in an increased sensitivity of the PCR. Detection of RNA viruses by PCR is often hampered by the interference of organic materials on the efficacy of PCR (Konet et al., 2000).
Definition
| RT-PCR | - reverse transcriptase-polymerase chain reaction |
| GuSCN | - guanidine isothiocyanate |
| RT | - room temperature |
| ul | - microliter |
Principle
This procedure combines several steps each reducing the interference of organic materials on the efficacy of PCR. Initially the material is treated with guanidine isothiocyanate (GuSCN) to avoid RNAse activity and to inactivate virus in the sample. Organ materials are extracted with acid phenol (for RNA viruses, here classical swine fever) and 1-Bromo-3-chloropropan. The nucleic acids are subsequently isolated by binding to acid-washed silica particles (Boom et al., 1990). Following washes in GuSCN/citrate and ethanol, nucleic acids are eluted in RNAse free water and are now ready for the RT-PCR procedure.
All procedures in this description must be performed in a flow-hood. GuSCN : Xn; R20/21/22, R 32 Phenol : Tx, C; R24/25, R34 1-Bromo-3-chloropropan (C3H6BrCl): Xn; R 22, R 40
Any virus will be inactivated by the high molar GuSCN used. However, to reduce the risk of contamination the procedure is performed in a clean area, i.e. PCR lab outside (mastermix extraction and preparation area). Changing of clothes and showering before entering the lab is required. All waste materials are incinerated immediately.
Required Materials
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Equipment
- Laminar air flow hood , Must be approved for work with 1-Bromo-3-chloropropan and phenol.
- Gloves
- Pipette
- Filter pipette tips
- Positive Displacement Pipettes
- Eppendorf tubes
- PCR tube racks
- Mini centrifuge
- Heating block
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Reagents
- Guanidine thiocyanate
- Distilled water
- Citric acid monohydrate
- Sodium Citrate Dihydrate
- Sterile water , Autoclaved or filtered
- RNase free water , DEPC
- Stop RNase Inhibitor
- EtOH
- Milli-Q water
Instructions
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Solutions
GuSCN solution (5M)
- GuSCN, 591 g
- Distilled water up to 1 liter.
- Keep in a dark bottle, at RT.
Citric acid (1M ,pH 5,2)
- Citric acid monohydrate, 4.90 g.
- Sodiumcitrate dihydrate, 23.52 g.
- Sterile water up to 100 ml.
- Autoclave and store at +4°C.
GuSCN/citrate (working solution of GuSCN)
- 30 ml GuSCN solution (5M)
- 936 ul 1M citric acid (pH 5.2)
- Store at +4°C.
Shelf life 1 month.
Elution buffer
- 600 ul nucleasefree water (DEPC)
- 20 ul PRIME RNase inhibitor.
70% EtOH
- 31.5 ml 99.9% EtOH
- 13.5 ml Milli-Q water
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Pretreatment of samples
Add 96 ?l serum to 304 ?l GuSCN/citrate, vortex well. At this stage the material may be stored at –20°C. If the material causes debris (organ suspensions, semen or full blood) centrifuge and transfer supernatant to clean tube.
-
Phenol/1-Bromo-3-chloropropan extraction of samples
Steps 2-4 are only relevant for organ materials.
- Thaw the samples, vortex and spin down, each tube is treated as follows:
- Add 40 ul of NaOAc.
- Add 400 ul of cold acid phenol; mix by turning the tubes several times.
- Add 120 ul 1-Bromo-3-chloropropan, use positive displacement pipette. Shake the tubes vigorously to mix the material.
- Spin the tubes at 11,300 g for 2 minutes.
- Transfer the supernatant (400 ul) to clean 1,5 ml Eppendorf tubes.
- The tubes may be frozen at this step or go immediately to Silica binding.
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Binding of RNA to Silica particles
- Add 2.5 ul Silica [AI-1.01.602] to each pretreated sample.
- Vortex briefly and leave the tubes for 20 min at RT on a rotating spear.
- Prepare washing solution, set the heating block for 65 °C, take 1 ml nuclease free Amresco water from the freezer (to be used in 3.10).
- Spin at 2400 x g for 20 seconds, remove the supernatant carefully using a pipette.
- Wash Silica twice using 475 ul GuSCN/citrate (vortex, spin 20 sec, 2400 g, and remove the supernatant by a pipette). It is essential that the Silica is 100% resuspended after each pelleting.
- Wash Silica twice using 475 ul 70% EtOH (vortex, spin 20 sec, 2400 g, and remove the supernatant by a pipette).
- Wash Silica once using 475 ul 99.9% EtOH (vortex, spin 20 sec, 2400 g, and remove the supernatant by a pipette).
- Spin the dried sample again (2400 g for 10 seconds) and remove as much supernatant as possible.
- Let the Silica dry for 5-10 minutes in the flow hood.
- Resuspend the pellet in 22.5 ul elution-buffer, vortex thoroughly to completely resuspend the Silica.
- Incubate the tube for 5 min at 65°C in the heating block.
- Spin tubes at 11,300 g for 2 min, transfer the supernatant to new Eppendorf tubes, the RNA samples are now ready for cDNA synthesis. Discard of the tubes containing pellets.
References
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Rapid and simple method for purification of nucleic acids ,
Author: R Boom, C J Sol, M M Salimans, C L Jansen, P M Wertheim-van Dillen, and J van der Noordaa | Date: March 1990 -
Inhibitors of RT-PCR in serum ,
Author: D.S. Konet, J.M.S. Mezencio, G. Babcock, F. Brown | Date: January 2000 -
Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen EL ,
Author: Uttenthal A, Storgaard T, Oleksiewicz MB, de Stricker K | Date: Apr 2003