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

Nadya G. Gurskaya, Luda Diatchenko, Alex Chenchik, Paul D. Siebert, George L. Khaspekov, Konstantin A. Lukyanov, Laura L. Vagner, Olga D. Ermolaeva, Sergey A. Lukyanov, Eugene D. Sverdlov

Research output: Contribution to journalArticlepeer-review

231 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)90-97
Number of pages8
JournalAnalytical Biochemistry
Volume240
Issue number1
DOIs
Publication statusPublished - 15 Aug 1996
Externally publishedYes

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