Extraction of Total RNA from Adipocytes

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 °C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12 - 30 µg total RNA from 2 × 10⁶ cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.

RNA Isolation - Adipocytes - CsCl Gradient Ultracentrifugation - Acid Phenol-Chloroform Extraction

References

• 1 Engeli S, Gorzelniak K, Kreutz R, Runkel N, Distler A, Sharma A M. Co-expression of renin-angiotensin system genes in human adipose tissue.  J Hypertens. 1999;  17 555-560
• 2 Gorzelniak K, Engeli S, Sharma A M. Standardizing the counting of adipocytes in cell culture.  Bio Techniques. 1998;  24 536-538
• 3 Glisin V, Crkvenjakov R, Byus C. Ribonucleic acid isolated by cesium chloride centrifugation.  Biochemistry. 1974;  13 2633-2637
• 4 Chirgwin J M, Przybyla A E, MacDonald R J, Rutter W J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.  Biochemistry. 1979;  18 5294-5299
• 5 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987;  162 156-159
• 6 Tavangar K, Hoffman A R, Kraemer F B. A micromethod for the isolation of total RNA from adipose tissue.  Anal Biochem. 1990;  186 60-63