title: Optimized T7 amplification system for microarray analysis.
abstract: Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).
authors.affiliation: Incyte Genomics, Palo Alto, CA 94304, USA.
authors.forename: C
authors.initials: C
authors.lastname: Pabón
authors.forename: Z
authors.initials: Z
authors.lastname: Modrusan
authors.forename: M V
authors.initials: MV
authors.lastname: Ruvolo
authors.forename: I M
authors.initials: IM
authors.lastname: Coleman
authors.forename: S
authors.initials: S
authors.lastname: Daniel
authors.forename: H
authors.initials: H
authors.lastname: Yue
authors.forename: L J
authors.initials: LJ
authors.lastname: Arnold
mesh_terms: D001709:Biotechnology; D012321:DNA-Directed RNA Polymerases; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D036103:Nanotechnology; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014764:Viral Proteins
chemical_list: D012333:RNA, Messenger; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases