Mplification efficiency and specificity (data not shown). Moreover, there had been reports that indicated that the Exiqon miRCURY assay, which also uses poly(A) tailing of the miRNAs, increased specificity utilizing LNA, but lead to a decrease in amplification efficiency [17,18]. To verify the amplification efficiency of the new assay, the amplification efficiencies of 10 miRNAs from SiHa cells were measured both with Exiqon miRCURY and the proposed assay. Amplification efficiency of the new assay (ranged from 90 to 105 ) was higher as compared with the Exiqon miRCURY method (85?5 ) (student’s t-test, P,0.05) (Table 1).heat-treated derived from an equal number of bone marrow cells. The consistency of the two sample preparation methods shed light on the applicability of this approach in handling precious samples.Assay Specificity and Cross-reactionThe sequences of the miRNA paralogs are identical except for 1? mismatched bases. To evaluate the specificity of our real-time PCR approach, we tested the amplification with primers having perfect complementarity or with 1? GSK -3203591 supplier nucleotides mismatched to the miR-32 sequence (Figure 4A). The primers with two or three mismatched bases did not result in products of significant levels (Figure 4B, mu2 and mu3), but primers with a single mismatched nucleotide led to almost the same extensive product amplification as perfectly matched primers (Figure 4B, mu1 and match). To modify the annealing efficient, we raised the PCR annealing temperature to 62uC, which is higher than the Tm value of single nucleotide mismatched primers. As a result, the modified PCR amplification level was reduced significantly (Figure 4B). These results attested that the specificity of the new miRNA quantification assay is favorable, but discrimination of miRNAs differing by a single base requires more stringent conditions. The specificity of the present assay was ulteriorly assessed with inherent miRNA family. The let-7 family is a representative miRNA family with members that have similar sequences. Crossreaction of five closely sequence-related members of the let-7 family (let-7a, let-7b, let-7c, let-7d and let-7e) differing in at least one nucleotides were employed for further analysis of the proposed approach (Figure 4C). Relative detection efficiency was calculated from differences of Cq between perfectly matched and mismatched targets, assuming 100 efficiency for the perfect match. The new assay displayed a high capability to discriminate between miRNA molecules, which differ by two or three bases (ranging from 0 to 0.3 ). Marginal cross reaction was observed mainly at miRNAs that differed by a single nucleotide with minute values ranging from 0.1 to 2.2 . Only the targeted miRNA was detected if more than three mismatched bases between any two miRNAs were present (Figure 4D). Most cross-reactions resulted from let-7a assay versus let-7c as a target. So the ML-281 forward primers of the PCR needed to meet special requirements to distinguish between let-7a and let-7c. These two forward primers have only one mismatched base at the 39-end. Therefore, to improve the specificity of the assay, the forward primers of the PCR were designed to contain mismatched base, and the PCR annealing temperature was raised to 60uC.Effect of Double-stranded DNA on the AssayChen et al. had observed the constraint of binding doublestranded genomic DNA owing to the stem-loop structure. To verify this effect in our assay, 5 ng of SiHa cell genomic DNA wa.Mplification efficiency and specificity (data not shown). Moreover, there had been reports that indicated that the Exiqon miRCURY assay, which also uses poly(A) tailing of the miRNAs, increased specificity utilizing LNA, but lead to a decrease in amplification efficiency [17,18]. To verify the amplification efficiency of the new assay, the amplification efficiencies of 10 miRNAs from SiHa cells were measured both with Exiqon miRCURY and the proposed assay. Amplification efficiency of the new assay (ranged from 90 to 105 ) was higher as compared with the Exiqon miRCURY method (85?5 ) (student’s t-test, P,0.05) (Table 1).heat-treated derived from an equal number of bone marrow cells. The consistency of the two sample preparation methods shed light on the applicability of this approach in handling precious samples.Assay Specificity and Cross-reactionThe sequences of the miRNA paralogs are identical except for 1? mismatched bases. To evaluate the specificity of our real-time PCR approach, we tested the amplification with primers having perfect complementarity or with 1? nucleotides mismatched to the miR-32 sequence (Figure 4A). The primers with two or three mismatched bases did not result in products of significant levels (Figure 4B, mu2 and mu3), but primers with a single mismatched nucleotide led to almost the same extensive product amplification as perfectly matched primers (Figure 4B, mu1 and match). To modify the annealing efficient, we raised the PCR annealing temperature to 62uC, which is higher than the Tm value of single nucleotide mismatched primers. As a result, the modified PCR amplification level was reduced significantly (Figure 4B). These results attested that the specificity of the new miRNA quantification assay is favorable, but discrimination of miRNAs differing by a single base requires more stringent conditions. The specificity of the present assay was ulteriorly assessed with inherent miRNA family. The let-7 family is a representative miRNA family with members that have similar sequences. Crossreaction of five closely sequence-related members of the let-7 family (let-7a, let-7b, let-7c, let-7d and let-7e) differing in at least one nucleotides were employed for further analysis of the proposed approach (Figure 4C). Relative detection efficiency was calculated from differences of Cq between perfectly matched and mismatched targets, assuming 100 efficiency for the perfect match. The new assay displayed a high capability to discriminate between miRNA molecules, which differ by two or three bases (ranging from 0 to 0.3 ). Marginal cross reaction was observed mainly at miRNAs that differed by a single nucleotide with minute values ranging from 0.1 to 2.2 . Only the targeted miRNA was detected if more than three mismatched bases between any two miRNAs were present (Figure 4D). Most cross-reactions resulted from let-7a assay versus let-7c as a target. So the forward primers of the PCR needed to meet special requirements to distinguish between let-7a and let-7c. These two forward primers have only one mismatched base at the 39-end. Therefore, to improve the specificity of the assay, the forward primers of the PCR were designed to contain mismatched base, and the PCR annealing temperature was raised to 60uC.Effect of Double-stranded DNA on the AssayChen et al. had observed the constraint of binding doublestranded genomic DNA owing to the stem-loop structure. To verify this effect in our assay, 5 ng of SiHa cell genomic DNA wa.