Figure 1
High overall consistency of log-ratio measurements across a wide range of transcript abundance. Normalized Cy3 (white), Cy5 (black), and log2(Cy5/Cy3) (grey) intensities from triplicate self-hybridization experiments for (A) Six genes with average intensities representing a 32-fold range (I = MAL7P1.89, II = PF14_0419, III = PF11_0528, IV = PF07_0118, V = MAL8P1.139, VI = PF11_0506) and (B) the 18 probes targeting MAL8P1.139 along with the probe average. (Error bars represent SEM). Non-feature background levels for the 8x15K arrays are very low with 98.6 % of probes yielding both Cy5 and Cy3 signals at least 2.5 standard deviations above background (data not shown). This needs to be taken into account during analysis, as weak non-specific binding of features targeting genes known to not be expressed in some samples occasionally produce very low signal that is nevertheless above non-feature background. In order to assess the minimum amounts of total RNA starting material and dye-labelled cDNA required, cDNA was generated from a pool of total RNA harvested at various stages of the IDC. As little as 500 ng of total RNA starting material yielded 460 ± 13 ng of amino-allyl labelled cDNA and 241 ± 4 ng of dye-coupled cDNA (Additional file 5) with 24 ± 0.4 fmol/ng of dye-incorporation. To test the minimum amount of cDNA required for hybridization, two separate pools of cDNAs with Cy3 and Cy5 were labelled respectively and hybridized a diminishing series of equal amounts from each pool (1,000 ng, 500 ng, 250 ng, 100 ng, 50 ng). Again examining the same set of genes representing a wide abundance range (see Figure 1A), only small differences in the Cy3/Cy5 log ratio measurements was observed across this range of hybridized material with the maximum fold-difference between any two of the 15 measurements for a given gene being 1.35-fold (Figure 2). No significant change in the number of transcripts with signal intensities called as “well above background” by Agilent Feature Extractor Software was observed when hybridizing decreasing amounts material (Additional file 6A). Furthermore, both Cy5 and Cy3 gene signal intensities array-wide correlated very highly (r > 0.96, Additional file 6B) across the dilution series down to 100 ng, and even at 50 ng of hybridized material gene signal intensities matched the 1000ng sample closely (r > 0.82, Additional file 6B). Thus, the amount of dye-coupled cDNA obtained from as little as 500 ng of total RNA is sufficient for performing two to four hybridization experiments.