title: Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis.
abstract: Gene expression analysis has become an invaluable tool for understanding gene function and regulation. However, global expression analysis requires large RNA quantities or RNA preamplification. We describe an isothermal messenger RNA (mRNA) amplification method, Ribo-SPIA, which generates micrograms of labeled cDNA from 5 ng of total RNA in 1 day for analysis on arrays or by PCR quantification. Highly reproducible GeneChip array performance (R2 > 0.95) was achieved with independent reactions starting with 5-100 ng Universal Human Reference total RNA. Targets prepared by the Ribo-SPIA procedure (20 ng total RNA input) or the Affymetrix Standard Protocol (10 microg total RNA) perform similarly, as indicated by gene call concordance (86%) and good correlation of differential gene expression determination (R2 = 0.82). Accuracy of transcript representation in cDNA generated by the Ribo-SPIA procedure was also demonstrated by PCR quantification of 33 transcripts, comparing differential expression in amplified and nonamplified cDNA (R2 = 0.97 over a range of nearly 10(6) infold change). Thus Ribo-SPIA amplification of mRNA is rapid, robust, highly accurate and reproducible, and sensitive enough to allow quantification of very low abundance transcripts.
authors.affiliation: NuGEN Technologies, San Carlos, CA 94070, USA. adafforn@nugeninc.com
authors.forename: Alan
authors.initials: A
authors.lastname: Dafforn
authors.forename: Pengchin
authors.initials: P
authors.lastname: Chen
authors.forename: Glenn
authors.initials: G
authors.lastname: Deng
authors.forename: Michael
authors.initials: M
authors.lastname: Herrler
authors.forename: Dawn
authors.initials: D
authors.lastname: Iglehart
authors.forename: Sriveda
authors.initials: S
authors.lastname: Koritala
authors.forename: Susan
authors.initials: S
authors.lastname: Lato
authors.forename: Susheela
authors.initials: S
authors.lastname: Pillarisetty
authors.forename: Reshma
authors.initials: R
authors.lastname: Purohit
authors.forename: Martin
authors.initials: M
authors.lastname: Wang
authors.forename: Shenglong
authors.initials: S
authors.lastname: Wang
authors.forename: Nurith
authors.initials: N
authors.lastname: Kurn
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D008832:Microchemistry / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D012333:RNA, Messenger