Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

• Published in: Clinical Chemistry
• Main Authors: Thormar, Hans G, Gudmundsson, Bjarki, Eiriksdottir, Freyja, Kil, Siyoen, Gunnarsson, Gudmundur H, Magnusson, Magnus Karl, Hsu, Jason C, Jonsson, Jon J
• Other Authors: Univ Iceland, Dept Biochem & Mol Biol, IS-101 Reykjavik, Iceland, Lifeind Ehf, Reykjavik, Iceland, Ohio State Univ, Dept Stat, Columbus, OH 43210 USA, Univ Iceland, Dept Pharmacol & Toxicol, IS-101 Reykjavik, Iceland, Landspitali Natl Univ Hosp, Dept Genet & Mol Med, Reykjavik, Iceland
• Format: Article in Journal/Newspaper
• Language: English
• Published: Amer Assoc Clinical Chemistry 2013
• Subjects: DNA kjarnsýra; DNA; Complementary; Electrophoresis; Gel; Two-Dimensional; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Iceland
• Online Access: http://hdl.handle.net/2336/324522; https://doi.org/10.1373/clinchem.2012.193839

To access publisher's full text version of this article click on the hyperlink at the bottom of the page The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. Technical Development Fund at Rannis/110473-0612 Icelandic Center for Research Funds University of Iceland Research Fund Landspitali Research Fund