1. Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect
- Author
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Luda Diatchenko, Konstantin A. Lukyanov, Alex Chenchik, Sergey Lukyanov, Amulia Nanisetti, Paul D. Siebert, Natalia Usman, and Mikhail V. Matz
- Subjects
Electrophoresis, Agar Gel ,DNA, Complementary ,cDNA library ,Total rna ,Biophysics ,RNA ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Polymerase Chain Reaction ,Rapid amplification of cDNA ends ,Complementary DNA ,Humans ,Primer (molecular biology) ,Muscle, Skeletal ,Molecular Biology ,Gene Library - Abstract
Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A)− RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.
- Published
- 1997