cDNA library preparation protocols distort miRNA research. a Comparison of change in miRNA level between wild-type and Dicer KO DLD cells obtained in Illumina (x axis) and HD samples (y axis). R2 = 0.62. b Number of known miRNAs found in DLD cells at different thresholds using Illumina or HD adapters. Regardless of chosen threshold, HD adapters identify more miRNAs. c Absolute quantification of eight known miRNAs (let-7i, miR-10a, miR-19b, miR-21, miR-25, miR-29b, miR-93, miR-375) obtained by Northern blot compared with number of times these miRNAs were sequenced using Illumina or HD adapters in DLD cell line. Data obtained with HD adapters correlates better with absolute quantifications (R2 = 0.70) than Illumina data (R2 = 0.12). d Number of PubMed citations and number of reads per experiment (data obtained from miRbase v17) of miRNAs conserved between mouse and human. MiRNAs with higher number of reads tend to be more extensively studied [R2 = 0.58, p-value < 10(−15)]. e-f Distributions of minimum free energy (MFE) of known human miRNAs concatenated with 5’ and 3’ adapter sequences. Using Illumina adapter sequences sRNA cloning kit V1.5 the set of miRNAs found by Illumina has lower average MFE than the set of miRNAs found by 454 (Wilcoxon test p = 0.01). We found the same result using the 3' adapter from sRNA cloning kit V1.0 (data not shown). e Conversely, using 454 adapter sequences average MFE is lower for set of miRNAs found by 454 (p = 0.07). f Analogous results for concatenation of miRNA only with 3’ adapter display a similar trend (see Additional file 5: Figure S7).