Verification of the specificity and sensitivity of RAKE. A) (a) A prototype small microarray designed to detect a limited number of miRNAs was first used to monitor the specificity of miRNA expression in HeLa and Jurkat cells. For purposes of verifying internal reproducibility of hybridization, each probe on the microarray slide was printed 4 times (spots 1, 2, 3 and 4 labeled at top of each column). The identity of individual probe is labeled next to the slide. Red left-hand filled circle indicates the miRNA expected to be expressed in HeLa cells. Red right-hand filled circle indicates the miRNA expected to be expressed in Jurkat cells. Red fully-filled circle indicates the miRNA expected to be present in both HeLa and Jurkat cells. Unfilled circle indicates the miRNA not expected to appear in either HeLa or Jurkat cells. Orange fully-filled circle represents "spike-in" oligos included act as positive controls to monitor successful hybridization performance. (b) We hybridized small RNAs isolated from HeLa (Left panel) and Jurkat cells (right panel) using microarray slides described in (a). Signals appear as green dots (fluorescence at 532 nm). With the exception of hsa-miR-142-3p in Jurkat cells, cell-specific signals were observed in the microarray hybridizations in patterns consistent with that expected for HeLa and Jurkat cells. 10-5 M of "spike-in" oligo (ath-miR-157a) was included in the experiment as an indicator of the maximum saturating signal from RAKE (saturated signals appear in white dots). Data are presented here in raw form without further modification or normalization. B) We demonstrated the specificity of RAKE by hybridizing small RNA isolated from Jurkat cells to a subset of polymorphic miRNA (hsa-let-7 family). The names, sequences of the miRNAs and the raw signals detected from the RAKE assay are listed. hsa-let-7c and hsa-let-7f differ from hsa-let-7a in one nucleotide base (highlighted in yellow). However, the signals detected for hsa-let-7a are approximately 16 times less than that detected for hsa-let-7c and hsa-let-7f, suggesting that RAKE can distinguish a single base difference. Similarly, the signals detected for hsa-let-7c are approximately 2.5 times higher than that detected in hsa-let-7b which has only one nucleotide difference (highlighted in yellow). C) To estimate the sensitivity of the RAKE assay, different concentrations of "spike-in" oligo (ath-miR-157a) were hybridized to the small microarray described in (a). The raw data from the four different hybridization reactions (each measuring four replicated spots) are presented on the X-axis at the indicated concentrations of "spike-in" target oligo. Signal intensity of each spot (median pixel) was measured and converted into log2 scale. A linear range of detection can be observed when the log2 values are plotted against the concentration of the "spike-in" oligos between 10-8 to 10-6 M. An approximate minimum detectable concentration in this RAKE assay is 10-7 M. Error bars represent the standard deviation of the values from the four replicated spottings of each probe.