Silica-based RNA Extraction (Step-by-Step Protocol)
Nucleic acid extraction is achieved with reagents such as detergents; these are added to a buffer to help make holes in the cell membrane (permeabilises the membrane). This permeabilisation allows the contents of the cell to spill out.
The next steps are to remove the protein fractions of the cell. The process includes addition of Proteases (proteases are enzymes that degrade proteins), as well as heating the lysate so that the proteins denature and become single threads of amino acid. In this process, reagents such as chloroform may be added for what is termed a liquid phase extraction of the nucleic acid (DNA/RNA).
An alternative and safer method is to use what is termed a solid phase extraction. Here the DNA after lysing the cell, is bound to a solid support such as silica or magnetic beads. Silica-based nucleic acid purification methods use a bind-wash-elute process. Nucleic acids bind to the silica membrane in the presence of chaotropic salts. Polysaccharides and proteins do not bind well to the column and residual traces are removed during the alcohol-based wash steps, along with the salts. This is the principle used by the PureLink kit (Catalog number: 12183018A, sufficient reagents to perform 50 preparations; column capacity is 1mg).
Needed reagents not provided by the Kit
2M DTT prep. for addition to the lysis buffer or you can use beta-mercaptoethanol. For this you need to know the mw. of DTT: 154.25g/mol (1.2mL (enough for 5 samples)). Weigh 0.370g and put into 1.2mL of nuclease-free water for 2M DTT. You can prepare these ahead of time and freeze them at -20oC. Add 20ul of 2M DTT to every 1mL of lysis buffer (final conc. is 40mM). B-mercaptoethanol may also be used but it is smelly and requires a fume hood when handling.
100% Ethanol
1. Lysis and Homogenization
Transfer ≤0.2 mL of whole blood into a 1.5 mL RNase-free microcentrifuge tube.
Add 0.2 mL of the prepared Lysis Buffer (containing 2-mercaptoethanol or 40mM DTT).
Vortex thoroughly to disrupt and lyse the cells, then centrifuge the lysate at 12,000 × g for 2 minutes at room temperature.
Carefully transfer the supernatant to a new 1.5 mL RNase-free microcentrifuge tube.
2. RNA Binding
Add 200 μL of 100% ethanol to the tube.
Disperse any precipitate by vortexing or pipetting up and down several times.
Transfer the sample (including any precipitate) to the Spin Cartridge.
Centrifuge at 12,000 × g for 15 seconds at room temperature and discard the flow-through.
(Optional: If DNA-free RNA is required, you may perform the on-column PureLink® DNase treatment at this stage).
On-Column PureLink® DNase Treatment Protocol
This is the most convenient method as it removes DNA while the RNA is bound to the Spin Cartridge, eliminating the need for additional clean-up steps later.
Preparation:
• Resuspension: Dissolve the lyophilized PureLink® DNase in 550 μL RNase-Free Water.
• DNase Mixture: For each sample, prepare an 80 μL mixture in a clean tube consisting of:
◦ 10X DNase I Reaction Buffer: 8 μL
◦ Resuspended DNase (~3U/μL): 10 μL
◦ RNase-Free Water: 62 μL
Procedure (to be performed after binding RNA to the cartridge):
1. Add 350 μL Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 × g for 15 seconds. Discard the flow-through and collection tube, then place the cartridge in a new collection tube.
2. Apply 80 μL of the prepared DNase mixture directly onto the surface of the Spin Cartridge membrane.
3. Incubate at room temperature for 15 minutes.
4. Add another 350 μL Wash Buffer I to the cartridge. Centrifuge at 12,000 × g for 15 seconds. Discard the flow-through and collection tube, then place the cartridge in a new collection tube.
5. Wash and Elute: Proceed with the standard protocol (two washes with 500 μL Wash Buffer II, drying the membrane, and eluting with 30–100 μL RNase-Free Water).
3. Washing
Add 700 μL Wash Buffer I to the Spin Cartridge.
Centrifuge at 12,000 × g for 15 seconds, discard the flow-through and the collection tube, and place the cartridge into a new collection tube.
Add 500 μL Wash Buffer II (prepared with ethanol) to the cartridge.
Centrifuge at 12,000 × g for 15 seconds and discard the flow-through.
Repeat the Wash Buffer II step once more.
Centrifuge the Spin Cartridge at 12,000 × g for 1 minute to completely dry the membrane.
4. Elution
Discard the collection tube and insert the Spin Cartridge into a Recovery Tube.
Add 30 μL–100 μL (100ul) of RNase-Free Water (or up to 3 x 100 μL for sequential elutions) to the center of the cartridge. I typically do 50ul.
Incubate at room temperature for 1 minute.
Centrifuge for 2 minutes at ≥12,000 × g to elute the purified RNA.
5. Storage
Purified RNA should be stored on ice if used within a few hours. For long-term storage, it should be kept at –80°C.
History of the approach
1979: Samples are lysed and homogenized in the presence of guanidinium isothiocyanate, a chaotropic salt capable of protecting the RNA from endogenous RNases (Chirgwin et al., 1979). After homogenization, ethanol is added to the sample.
1979: The sample is then processed through a Spin Cartridge containing a clear silica-based membrane to which the RNA binds. Any impurities are effectively removed by
subsequent washing (Vogelstein & Gillespie, 1979).
The purified total RNA is then eluted in RNase-Free Water (or Tris Buffer, pH 7.5) and may be used for:
• Real-time-PCR (RT-PCR)
• Real-time quantitative–PCR (qRT–PCR)
• Northern blotting
• Nuclease protection assays
• RNA amplification for microarray analysis
• cDNA library preparation after poly(A)+ selection
Related posts
How To Remove Globin and rRNA From Blood-based RNA samples: A Complete Guide: https://adwoabiotech.blogspot.com/2026/03/globin-rna-ribosomal-depletion-protocol-blood.html
DEPC and RNA What It Is and Why It Matters: https://adwoabiotech.blogspot.com/2025/06/depc-and-rna-what-it-is-and-why-it.html
References
Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. Z. (1979) Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonucleases. Biochem. 18, 5294-5299
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Plainview, New York
Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615-619
Wilfinger, W. W., Mackey, K., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474-476, 478-481
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