Rapid RNA isolation without the use of commercial kits: application to small tissue samples.
We describe an adapted version of the Chomczynski and Sacchi [Anal Biochem (1987) 162:156-159] RNA isolation procedure that can be performed in less than 1 h on small (<15 mg) tissue samples, using commonly available reagents. Our modifications included: (1) one rather than two precipitation steps in the aqueous phase with 99% ethanol and (2) elimination of the 1-h incubation step at -20 degrees C. Our adaptations resulted in RNA yield (microg/mg of tissue) and purity (260/280 nm ratios) comparable to those of the original procedure. Furthermore, the isolated RNA was successfully utilized in reverse transcriptase-polymerase chain reaction assays, suggesting that it was essentially free of carry-over contaminants that could inhibit enzymatic reactions. When tested on tissue sample sizes of 7-12 mg, our adapted procedure allowed the recovery of enough total RNA for use in techniques such as Northern blot analysis. Our modified technique is therefore an inexpensive alternative to commercially available kits when isolating good-quality RNA from very small tissue samples, such as those obtained from needle biopsies.
Gauthier
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ER
Department of Chemistry and Biochemistry, Laurentian University, Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6.
Madison
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SD
Michel
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RN
D000818:Animals; D001923:Brain Chemistry; D004064:Digestive System / Q000737:chemistry; D005260:Female; D005821:Genetic Techniques; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013154:Spleen / Q000737:chemistry
D012313:RNA