Big results from small samples: Evaluation of amplification protocols for gene expression profiling

Agnes Viale, Juan Li, Jay Tiesman, Susan Hester, Aldo Massimi, Chandi Griffin, George Grills, Greg Khitrov, Kathryn Lilley, Kevin Knudtson, Bill Ward, Karl Kornacker, Chin Yi Chu, Herbert Auer, Andrew I. Brooks

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RNA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RNA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.

Original languageEnglish (US)
Pages (from-to)150-161
Number of pages12
JournalJournal of Biomolecular Techniques
Volume18
Issue number3
StatePublished - Jul 1 2007

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Keywords

  • Gene expression
  • GeneChip
  • Microarray
  • Quantitative PCR
  • RNA amplification
  • Small sample

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Viale, A., Li, J., Tiesman, J., Hester, S., Massimi, A., Griffin, C., Grills, G., Khitrov, G., Lilley, K., Knudtson, K., Ward, B., Kornacker, K., Chu, C. Y., Auer, H., & Brooks, A. I. (2007). Big results from small samples: Evaluation of amplification protocols for gene expression profiling. Journal of Biomolecular Techniques, 18(3), 150-161.