Open AccessReverse Transcription in the Presence of Dideoxynucleotides to Increase the Sensitivity of Expression Monitoring with cDNA Arrays
Author(s) -
Charles Decraene,
Isabelle Reguigne-Arnould,
Charles Auffray,
Geneviève Piétu
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99275st03
Subject(s) - complementary dna , nucleic acid , reverse transcriptase , microbiology and biotechnology , biology , oligonucleotide , cdna library , reverse transcription polymerase chain reaction , transcription (linguistics) , messenger rna , rna , dna , gene , genetics , linguistics , philosophy
Complex cDNA targets conforming to the nomenclature proposed by Duggan et al. (Nat. Genet. 21 [1 Suppl.]:10–14) (where the immobilized nucleic acid is called “probe” and free nucleic acid is called “target”) were prepared by priming reverse transcription of mRNA with oligo-(dT)-primers or random oligonucleotide primers in the presence or absence of dideoxynucleotides. These targets were then hybridized to high-density filters spotted with 1339 cDNA clone inserts from a skeletal muscle library. The addition of dideoxynucleotides was found to significantly improve the efficiency and reproducibility of the reverse transcription reaction, and consequently, to improve detection of the least abundant transcripts in expression profiling experiments.