Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 the MIQE guidelines, accuracy of a RT-qPCR refers to the difference between experimentally measured and actual concentrations as fold difference [16]. Accordingly, we per- formed miR-Q arrays for all concentrations and deter- mined the fold differences between 3.3 and 33 pM as well as 33 and 330 pM applying the ΔΔCq algorithm, respec- tively. The reactions revealed 12 fold difference of both let- 7e and miR-143 and 20 fold difference of miR-145 between 3.3 and 33 pM spike-in (Figure 4B). While detection of all other miRNAs remained unaffected, in the 33 pM spiked sample there was a slight cross reaction between let-7e spike-in and other let-7 family members resulting in 1.3 to 1.9 fold difference compared to the 3.3 pM control. Higher dosage of spike-in however led to a higher cross reaction within the let-7 family. As shown in Figure 4C, let-7e spike-in at a final concentration of 330 pM created 5 fold increased detection of let-7a and 6.5 fold detection of the specific target let-7e compared to the 33 pM spiked sam- ple. Furthermore, there was a 22.4 fold difference of miR- 143 and a 34 fold difference of miR-145 between both sam- ples (Figure 4C). As we discussed earlier, the higher the concentration of the spike-in, the higher is the cross reac- tion between the assays and paralogous targets of a miRNA family. These data verify the accuracy of miR-Q arrays especially at low spike-in concentrations, which rather represent cellular and physiological miRNA concen- trations compared to higher concentrations producing increased cross reactivity. We have shown earlier that the miR-Q approach is able to accurately discriminate between the let-7 family mem- bers [9] pointing to the analytical specificity of this approach. Since many miRNA families exist and their members often possess few nucleotide exchanges, we examined the discriminative power of miR-Q by studying the intra-family-interaction exemplified by miR-29 and miR-30 family members. The cross reaction between the specific miR-Q and a paralogous miRNA target was determined along the entire dynamic range of assays. It was intriguing that a two nucleotide mismatch within the miRNA specific part of a ‘miRNA’-rev oligonucleotide and a paralogous miRNA target resulted in less than 0.25% cross reactivity of assays to false targets. When the non-specific target differed only in one nucleotide the cross reaction varied between 10 and 20% (Figure 5A and 5B). This observation was true for the miR-29 as well as miR-30 family. Although the determined values point out the high target-specificity of miR-Q assays, in one mis- match cases it has to be considered that around 10% of the measured expression value may be derived from paralogous miRNAs of a family. Since the ‘miRNA’-rev oligonucleotides (table 1) confer the specificity of miR-Q assays; the respective design determines the magnitude of cross reactivity and the analytical specificity. For mini- mising cross reactivity of miR-Q assays and if possible, Page 7 of 11 Figure 5 Analytical specificity of miR-Q arrays. Figure 5A shows the alignment of miR-29 as well as miR-30 family members. Dashed borders indicate binding sites of ‘miRNA’-rev primers. The percentage of cross reaction is given between assay-specific and unspecific miRNA targets. Assay-specific targets represent the full value (Figure 5B). The design of the miR-Q approach allows simultaneous quantification of a canonical miRNA as well as its isomiRs as exemplified by miR-24 (Figure 5C). The columns in Figure 5C show triplicate determination of the linear range using synthetic miR-24 as well as synthetic isomiRs. Pearson correlation analysis revealed high linearity between all data sets possessing r < 0.9969 and two-tailed P values < 0.0001 (Figure 5D).