title: Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Biochemistry and Molecular Biology, University of Iceland, Reykjavik, Iceland.
authors: Hans G
authors: HG
authors: Thormar
authors: Bjarki
authors: B
authors: Gudmundsson
authors: Freyja
authors: F
authors: Eiriksdottir
authors: Siyoen
authors: S
authors: Kil
authors: Gudmundur H
authors: GH
authors: Gunnarsson
authors: Magnus Karl
authors: MK
authors: Magnusson
authors: Jason C
authors: JC
authors: Hsu
authors: Jon J
authors: JJ
authors: Jonsson
mesh_terms: D004247:DNA / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results
chemical_list: D018076:DNA, Complementary; D004247:DNA