Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Feb 25;16(4):1487–1497. doi: 10.1093/nar/16.4.1487

Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis.

H C Birnboim 1
PMCID: PMC336329  PMID: 2450333

Abstract

The study of messenger RNA in mammalian cells by Northern analysis requires the extraction of intact RNA in pure form. Although a number of reliable techniques have been developed for the purpose, most are fairly complex, involving steps such as ultracentrifugation and multiple extractions with large volumes of phenol and chloroform. When the number of cell samples to be analyzed is large, these techniques can be unwieldy. I now describe an RNA purification procedure which is simple enough to allow handling of a large number of cultured cell samples. It uses safe and inexpensive reagents and produces a high yield of pure total cell RNA, essentially free of DNA and ribonuclease, suitable for Northern analysis. The procedure also allows extraction of intact RNA from human granulocytes, cells which are rich in ribonuclease and contain very low amounts of RNA.

Full text

PDF
1491

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C. DNA strand breakage in human leukocytes exposed to a tumor promoter, phorbol myristate acetate. Science. 1982 Mar 5;215(4537):1247–1249. doi: 10.1126/science.6276978. [DOI] [PubMed] [Google Scholar]
  4. Cathala G., Savouret J. F., Mendez B., West B. L., Karin M., Martial J. A., Baxter J. D. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
  5. 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 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Feramisco J. R., Smart J. E., Burridge K., Helfman D. M., Thomas G. P. Co-existence of vinculin and a vinculin-like protein of higher molecular weight in smooth muscle. J Biol Chem. 1982 Sep 25;257(18):11024–11031. [PubMed] [Google Scholar]
  8. Hamada H., Petrino M. G., Kakunaga T. Molecular structure and evolutionary origin of human cardiac muscle actin gene. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5901–5905. doi: 10.1073/pnas.79.19.5901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Krawetz S. A., States J. C., Dixon G. H. Isolation and fractionation of total nucleic acids from tissues and cells. J Biochem Biophys Methods. 1986 Jan;12(1-2):29–36. doi: 10.1016/0165-022x(86)90048-5. [DOI] [PubMed] [Google Scholar]
  10. La Fleur M., Beaulieu A. D., Kreis C., Poubelle P. Fibronectin gene expression in polymorphonuclear leukocytes. Accumulation of mRNA in inflammatory cells. J Biol Chem. 1987 Feb 15;262(5):2111–2115. [PubMed] [Google Scholar]
  11. TODARO G. J., GREEN H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963 May;17:299–313. doi: 10.1083/jcb.17.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Yang A., Miller D. M. Purification of functional RNA from human granulocytes. J Lab Clin Med. 1985 Jan;105(1):94–98. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES