Evaluation of vector-primed cDNA library production from microgram quantities of total RNA

Nucleic Acids Res. 2004 Dec 15;32(22):e183. doi: 10.1093/nar/gnh181.

Abstract

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • DNA, Complementary / biosynthesis*
  • DNA, Complementary / genetics
  • Gene Library*
  • Genetic Vectors*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • RNA / metabolism
  • Sequence Analysis, DNA

Substances

  • DNA, Complementary
  • RNA