miRNA Analysis and Non-Coding RNA Support—Troubleshooting

The recommended input range for the TaqMan™ MicroRNA Assays is 1-10 ng of total RNA. However, some targets may not be as abundant as others and require more input. You can try titrating the amount of input for your assay, up to 250 ng of total RNA. If this does not help, you can also try doubling the amount of RT enzyme to 6.6 U/µL (assuming that the target of interest is present). Please go here for additional troubleshooting help.

Every assay is tested for NTC. We guarantee an NTC C_t of ≥38 for the microRNA assays. This is a (–RT) NTC, meaning a test with RT primer but without RT enzyme and RNA (water is used instead of RNA).

If you are seeing amplification with a Ct of <38 with an assay in a NTC well, try changing your reagents. Take extra precautions if you are working with any plasmids or other artificial templates in the lab, as they can easily contaminate reagents and surfaces and are very difficult to eliminate. You may have to test by working in a different location with different pipets and reagents. You can also clean surfaces with a DNA degradation solution like DNAZap™ PCR DNA Degradation Solutions.

Megaplex™ RT and PreAmp Primers are highly multiplexed pools designed to significantly streamline the workflow when profiling many miRNA targets in a single experiment. When combined in a large multiplex pool, a small subset of assays exhibit lower no-template control (NTC) Ct values (<35) than when run individually. This is not an assay design issue.

In the interest of providing the broadest coverage possible, customers have requested that we include these assays on the profiling array, but with the expectation that they are to be considered semi-quantitative. As a result, fold change measurements using these assays may be less than the true value. To aid data interpretation, the identity of these assays is provided in the Megaplex™ Assay Performance File. If accurate fold change measurements for these assays is necessary, it is recommended that changes detected using the Megaplex™ workflow be validated using the corresponding individual TaqMan™ MicroRNA Assay.

The TaqMan™ miRNA array cards allow one to profile over 300 different miRNA targets on one card. Due to the large number of targets and the nature of your sample, it is possible that many miRNA on the card will not be expressed on your sample, or expressed at very low levels. This is one reason why you will not see good amplification plots for every spot on the array card. Another possibility is the sample quality or input. Make sure to check the concentration and quality of the RNA sample before reverse transcription. If you have less than 350 ng of total RNA, then we recommend using preamplification. Check the C_t values for U6 on the card, and see if it is in the expected range for your typical samples.