Discrimination between miRNAs with single nucleotide differences. A Position of the single nucleotide mismatches relative to the PCR primers for the ssc-let-7a, ssc-miR-23a, ssc-miR-125b and ssc-miR-150 qPCR assays. The ssc-miR-23b sequence used for mismatch discrimination was taken from miRBase and is different from the ssc-miR-23b sequence found in uterus and used for designing the ssc-miR-23b qPCR primers (Table 1). B Discrimination between closely related miRNA templates for miR-specific qPCR assays with DNA primers. Mismatches in the miRNA compared to the PCR primers are underlined. The data represents the results of three to four measurements. C Amplification curves of ssc-let-7a and ssc-let-7e synthetic template in the ssc-let-7a miR-specific qPCR assays. All samples including the no template control (ntc) contained a final concentration of 0.2 ng/μl salmon sperm DNA.