Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate.
The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.
Gurskaya
N G
NG
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, ul. Miklukho-Maklaya 16/10, Moscow, 117871, Russia.
Diatchenko
L
L
Chenchik
A
A
Siebert
P D
PD
Khaspekov
G L
GL
Lukyanov
K A
KA
Vagner
L L
LL
Ermolaeva
O D
OD
Lukyanov
S A
SA
Sverdlov
E D
ED
D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D015698:Genomic Library; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects*; D010835:Phytohemagglutinins / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012984:Software; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation
D018076:DNA, Complementary; D010835:Phytohemagglutinins; D013755:Tetradecanoylphorbol Acetate