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
Van Gelder RN. von Zastrow ME. Yool A. Dement WC. Barchas JD.
Amplified RNA synthesized from limited quantities of heterogeneous
Proceedings of the National Academy of Sciences of the United States
America. 87(5):1663-7, 1990 Mar.
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
nucleic acids sufficient for use in standard cloning strategies, we
devised a method for producing amplified heterogeneous populations of
from limited quantities of cDNA. Whole cerebellar RNA was primed with
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
(aRNA). Up to 80-fold molar amplification has been achieved from
quantities of cDNA. The amplified material is similar in size
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
(G-protein) alpha subunits have been detected in the amplified
By using in situ transcription to generate cDNA, sequences for
have been detected in aRNA derived from single cerebellar tissue
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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