Validation of cDNA microarray gene expression data obtained from linearly amplified RNA

Abstract

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.

Figure 1

Linear RNA amplification. (A) Various amounts of total RNA isolated from the SUDHL-6 cell line were used for two rounds of linear amplification. With decreasing template concentration below approximately 500 ng, the amplified RNA product shows a significant decline in both quantity and quality (size). Lane 1, RNA ladder; lane 2, 1000 ng starting RNA template; lane 3, 500 ng starting RNA template; lane 4, 100 ng starting RNA template; lane 5, 10 ng starting RNA template; lane 6, 1 ng starting RNA template; lane 7, 0.1 ng starting RNA template; lane 8, DNA ladder. (B) Aliquots of 1000 ng of total RNA from the SUDHL-6 cell line were subjected to two rounds of RNA amplification. One round of amplification resulted in approximately 15-fold amplification and two rounds showed approximately 190-fold amplification. Lane 1, RNA ladder; lane 2, two rounds of amplification; lane 3, one round of amplification; lane 4, DNA ladder. (C) The quality and quantity of the amplified RNA is directly dependent upon the quality of the starting template RNA. Partially degraded RNA results in much lower amounts of amplified product and smaller product size after two rounds of linear amplication compared with amplified product obtained from intact starting material. Lane 1, RNA ladder; lane 2, two rounds of amplification starting from 1000 ng of intact total RNA; lane 3, two rounds of amplification starting from 1000 ng of partially degraded total RNA; lane 4, DNA ladder. (D) The concentration of the oligo-dT(15)-T7 promoter primer used to make the cDNA is a crucial component of the RNA amplification reaction. Template independent products (primer dimers) are produced when higher concentrations of oligo-dT(15)-T7 primer are used in the amplification reaction. Lane 1, RNA ladder; lane 2, 1000 ng oligo-dT(15)-T7 primer; lane 3, 500 ng oligo-dT(15)-T7 primer; lane 4, 100 ng oligo-dT(15)-T7 primer; lane 5, DNA ladder. (E) One round of amplification produced amplified RNA (aRNA) from SUDHL-1 (lanes 1 and 2) and Karpas 299 (lanes 3 and 4) starting from 2 μg total RNA template using the RiboAMP kit; lane 5, DNA ladder.

Figure 2

Representative scatter plots of log2 transformed data from cDNA microarray hybridisations. Equal amounts (2 μg) of either mRNA or amplified RNA (aRNA) were used for each comparison. (A) Scatter plots representing averaged cDNA microarray hybridisations comparing SUDHL-6 aRNA samples; r  =  0.9904. (B) Scatter plots representing averaged cDNA microarray hybridisations comparing SUDHL-6 aRNA and mRNA samples; r  =  0.9038. (C) Scatter plots representing averaged cDNA microarray hybridisations comparing SUDHL-6 mRNA to purified tonsillar B cell mRNA; r  =  0.8783. (D) Scatter plots representing averaged cDNA microarray hybridisations comparing SUDHL-6 aRNA to purified tonsillar B cell aRNA; r  =  0.9763.

Figure 3

Comparison of cDNA microarray expression data and quantitative fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) data for selected differentially expressed genes identified by microarray analysis of mRNA and amplified RNA (aRNA) samples obtained from the SUDHL-6 cell line. RT-PCR results were calculated as described in the Materials and Methods. We consistently found a larger dynamic range of expression for our RT-PCR data relative to the corresponding cDNA microarray expression data. Results are included for the potassium voltage gated channel shaker related subfamily β member 2 (KCNAB2), KIAA0246, DNA (cytosine-5)-methyltransferase 3α (DNMT3A), major histocompatibility complex (MHC) class II DMα (HLA-DMA), MHC class II DPβ1 (HLA-DPB1), junD, fosB, and CD79A antigen (immunoglobulin associated α) genes, and selected expressed sequence tags (EST1 and EST2).