Identification of differentially expressed genes from limited amounts of RNA.
The identification of cellular RNA expression profiles by differential display (DD) involves the visualization of RT-PCR products from the RNA. Traditionally, DD protocols require 200-500 ng RNA for each RT reaction. Thus, the limiting factor in DD is the amount of RNA available and the sensitivity of the RT reaction. By replacing the type of reverse transcriptase in our method, the sensitivity of DD increased up to 100-fold. Very significantly, the cDNA species obtained are higher in molecular weight, increasing the chances of detection of differential display genes with less background bands. The false positives and background in general also decreased due to the utilization of Taq polymerase antibody to facilitated DNA synthesis in the PCR reaction step. The reverse transcriptases described here may have a greater priming capacity as well as strong processivity which would explain the higher sensitivity accomplished in comparison to more standard reverse transcriptases. Additionally, the application of a more sensitive DD to samples when the amount of RNA is limited would be highly recommended.
Bosch
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Dana-Farber Cancer Institute Division of Cancer Biology and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA.
Melichar
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H
Pardee
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AB
D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D012313:RNA / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D012313:RNA; D012194:RNA-Directed DNA Polymerase