Custom TaqMan™ Small RNA Assay

The TaqMan miRNA asays are guaranteed to meet the following:
Dynamic Range: > 6 logs10 with > 0.97 linearity (R2 value)
Specificity: Majority of assays have < 5% cross reactivity with closely related sequences. NTC background: Ct > 38.0
Lot-to-Lot Reproducibility: Difference between Ct's < 0.6 Ct when different lots of an assay are run with the same sample and master mix from the same lot on the sample plate
Amplification Efficiency: ranging from 90-110% across 5 logs10

Please see the document “TaqMan Assays QPCR Guarantee Program” for more details.

If you are using an Applied Biosystems instrument, we recommend using Expression Suite to analyze data from the TaqMan MicroRNA Array Cards. After importing the .sds or .eds files, you can perform all the QC and data analysis within the tool. For more details on how to use Expression Suite software, please see this video series: https://learn.thermofisher.com/courses/view/id/325

It is well understood within the miRNA community that designing assays for miRNAs is challenging due to their short length (<22 bases) and closely related sequences. Although we have not tested cross reactivity of every closely related species, we have demonstrated that we can achieve <5% cross reactivity between a single nucleotide mismatch. Specificity of an assay depends on the number of mismatches to its closest homologue, the location of the mismatch, and the surrounding bases, making cross reactivity difficult to predict. As a general rule, the most difficult miRNA targets to discriminate are those with minimal mismatches localized to the 5' region of the sequence, and it is close to impossible to design an assay that discriminates between a single mismatch at the 5' most base. In addition, the assays in our catalog have been designed to provide a balance between specificity and sensitivity: an assay may be very specific but lack the needed sensitivity, or vice versa. To achieve this balance, and to ensure the highest sensitivity and to reduce false positives, TaqMan MicroRNA Assays must have an NTC background Ct > 38.0 and display good linearity across at least 3 logs10 (ideal R2 > 0.98).

Deep sequencing analysis of mature miRNAs revealed that many miRNAs have either an addition or deletion of 1-3 bases at the 3' and less frequently at the 5' terminal end. These are often referred to as isomiRs. The sequence deposited in miRBase is the canonical sequence derived by aligning sequences from current deep sequencing data. Thus far, there has been no biological relevance attached to these different forms since they exclusively occur outside the seed sequence. For that reason, the changes detected in the expression level of one isomer are proportional to changes within the entire pool. As a result, there may be a shift in raw Ct value using assays targeting two separate isomiRs. However, the relative expression ddCt has been demonstrated to be roughly the same. It should be noted that, although TaqMan MicroRNA Assays are designed to be sequence specific, they will detect a small spectrum of isomiRs. Depending on the number and composition at the 3' end, an assay may detect the +1 and +2 isomiRs but not the -1 or -2 forms.

Use multiple replicates and consider, when possible, using an overall study control that is used in every assay to monitor potential day-to-day/run-to-run/across study variability.