Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect.
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.
Lukyanov
K
K
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow.
Diatchenko
L
L
Chenchik
A
A
Nanisetti
A
A
Siebert
P
P
Usman
N
N
Matz
M
M
Lukyanov
S
S
D018076:DNA, Complementary / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA
D018076:DNA, Complementary; D012313:RNA