Multi-targeted priming for genome-wide gene expression assays

摘要

The present invention is a method of generating a library of expressed cDNA sequences using multi-targeted primers that are complementary to degenerate motifs present in a large proportion of corresponding mRNA sequences. Multi-targeting priming (MTP) for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences. Priming with MTPs in addition to oligo-dT can result in higher sensitivity, a greater number of genes whose expression is well measured, a greater number of genes whose differences in gene expression are detected to be statistically, and a greater power to detect meager differences in expression.

主权项

1. A method of generating a library of expressed cDNA sequences, comprising the steps of:
(1) providing one or more RNA sequences from a genome database of a selected organism; (2) identifying one or more degenerate motifs that bind to a large proportion of mRNA sequences, said one or more identified degenerate motifs being located at multiple sites within said one or more mRNA sequences, in the steps of:
randomly defining a degenerate oligonucleotide sequence;for the degenerate oligonucleotide sequence, calculating a score according to the formula:Score=mRNAhits#⁢⁢mRNAs*(1+tRNAhits+rRNAhits) where mRNA hits, tRNA hits, and rRNA hits are the number of hits of said degenerate oligonucleotide sequence to the annotated mRNAs, tRNAs, and rRNAs, respectively, and #mRNAs is the total number of annotated mRNAs;randomly mutating the degenerate oligonucleotide sequence;calculating the score for the randomly mutated degenerate oligonucleotide sequence;comparing the score for the randomly defined degenerate oligonucleotide sequence with the score for the randomly mutated degenerate oligonucleotide sequence; (3) based on the comparison, providing one or more multi-targeted primers that are complementary to one or more of said degenerate motifs; and (4) combining said multi-targeted primers with mRNA molecules obtained from the organism in a reverse transcription assay to generate a library of expressed cDNA sequences.