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ORIGINAL ARTICLE |
1 Department of Pathology, University of Utah Health Sciences Centre, Salt Lake City, UT 84132, USA
2 ARUP Institute for Clinical and Experimental Pathology, University of Utah Health Sciences Centre
3 Department of Pathology, Roger Williams Hospital, Providence, Rhode Island, 02908, USA
Correspondence to:
Dr K S J Elenitoba-Johnson
Division of Anatomic Pathology, University of Utah Health Sciences Centre, 50 North Medical Drive, Salt Lake City, UT 84132, USA; kojo.elenitobaj{at}path.utah.edu
Background: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail.
Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR).
Results: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template.
Conclusions: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.
Keywords: amplified RNA; in vitro transcription; microarray; expression profiling; real time PCR
Abbreviations: aRNA, amplified RNA; CT, crossing threshold; DLBCL, diffuse large B cell lymphoma; GAPDH, glyceraldehyde phosphate dehydrogenase; KCNAB2, potassium voltage gated channel shaker related subfamily ß member 2; DNMT3A, DNA (cytosine-5)-methyltransferase 3
; MHC, major histocompatibility complex; PCR, polymerase chain reaction; RT, reverse transcriptase
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