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.

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).

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.