Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans

Anal Biochem. 1993 May 1;210(2):337-43. doi: 10.1006/abio.1993.1205.

Abstract

RNA isolated by conventional guanidinium isothiocyanate methods from tissues of a mollusc (red abalone: Haliotis rufescens) is largely degraded and discolored by contaminants. These contaminants are associated with inhibition of reverse transcriptase, prevent accurate spectrophotometric determination of RNA concentration, and impart undesirable viscosity to the preparations. A cold two-step method of RNA isolation was devised which provides high yields of full-length RNA templates from these tissues and eliminates the discolored contaminant. Immediately following homogenization of tissues at ca. 5 degrees C, which proved crucial for the recovery of high-molecular weight species, the RNA is isolated from the bulk of the RNase by a single acid-phenol-chloroform extraction at 0 degrees C. The inhibitor of reverse transcriptase, suspected to be a proteoglycan (or a similar high-molecular-weight polyanion) component of the intestinal mucus, is eliminated only by a second purification step employing ultracentrifugation through a dense cushion of CsCl. This cold two-step method should prove useful for providing full-length RNA templates relatively free of polysaccharide, a common contaminant of RNA preparations, from both plant and animal tissues.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Avian Myeloblastosis Virus / enzymology
  • Base Sequence
  • Chemistry Techniques, Analytical / methods
  • DNA / biosynthesis
  • Guanidine
  • Guanidines
  • Intestines / chemistry
  • Intestines / enzymology
  • Molecular Sequence Data
  • Mollusca
  • Moloney murine leukemia virus / enzymology
  • Pancreas / chemistry
  • Pancreas / enzymology
  • Poly A / isolation & purification
  • Proteoglycans* / analysis
  • RNA / genetics*
  • RNA / isolation & purification*
  • RNA-Directed DNA Polymerase / genetics
  • Rats
  • Ribonucleases* / analysis
  • Templates, Genetic
  • Transcription, Genetic / genetics*
  • Ultracentrifugation

Substances

  • Guanidines
  • Proteoglycans
  • Poly A
  • RNA
  • DNA
  • RNA-Directed DNA Polymerase
  • Ribonucleases
  • Guanidine