Reverse Northern

Matthew P. Frosch, M.D., Ph.D. frosch at dsg.harvard.edu
Tue Sep 24 09:21:18 EST 1996


In order to find Jim Eberwine's paper, you need to spell his name
correctly!

Authors
  Van Gelder RN.  von Zastrow ME.  Yool A.  Dement WC.  Barchas JD. 
  Eberwine JH.
Title
  Amplified RNA synthesized from limited quantities of heterogeneous
cDNA.
Source
  Proceedings of the National Academy of Sciences of the United States
of
  America.  87(5):1663-7, 1990 Mar.
Abstract
  The heterogeneity of neural gene expression and the spatially limited
  expression of many low-abundance messenger RNAs in the brain has made
  cloning and analysis of such messages difficult. To generate amounts
of
  nucleic acids sufficient for use in standard cloning strategies, we
have
  devised a method for producing amplified heterogeneous populations of
RNA
  from limited quantities of cDNA. Whole cerebellar RNA was primed with
a
  synthetic oligonucleotide containing the T7 RNA polymerase promoter
  sequence 5' to a polythymidylate region. After second-strand cDNA
  synthesis, T7 RNA polymerase was used to generate amplified antisense
RNA
  (aRNA). Up to 80-fold molar amplification has been achieved from
nanogram
  quantities of cDNA. The amplified material is similar in size
distribution
  to the parent cDNA and shows sequence heterogeneity as assessed by
  Southern and Northern blot analysis. Specific messages for
  moderate-abundance mRNAs for actin and guanine nucleotide-binding
protein
  (G-protein) alpha subunits have been detected in the amplified
material.
  By using in situ transcription to generate cDNA, sequences for
cyclophilin
  have been detected in aRNA derived from single cerebellar tissue
sections.
  cDNA derived from a single cerebellar Purkinje cell also has been
  amplified and yields material that hybridizes to cognate whole RNA and
  mRNA but not to Escherichia coli RNA.



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