Analysis of Affymetrix GeneChip data using amplified RNA

Biotechniques. 2006 Feb;40(2):165-6, 168, 170. doi: 10.2144/000112057.

Abstract

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.

Publication types

  • Comparative Study

MeSH terms

  • Algorithms
  • DNA, Complementary / genetics
  • DNA-Directed RNA Polymerases
  • Microdissection
  • Nucleic Acid Amplification Techniques / methods*
  • Nucleic Acid Hybridization
  • Oligonucleotide Array Sequence Analysis / methods*
  • RNA / analysis*
  • RNA / genetics
  • Transcription, Genetic
  • Viral Proteins

Substances

  • DNA, Complementary
  • Viral Proteins
  • RNA
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases