miRNA and pri-miRNA Support—Troubleshooting
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TaqMan® MicroRNA Assays
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).
You can easily see all the content on the microRNA assays by downloading this list. You can then use the column headers to the right to sort by either human or rodent for the respective array content.
Every assay is tested for NTC. We guarantee an NTC Ct 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 get rid of. 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.
Yes. You can use the TaqMan® MicroRNA Assays for absolute quantitation when you need to know the exact number of copies being expressed. For this type of analysis, you will need to obtain a synthetic microRNA to create a standard curve. You can order a synthetic RNA oligo for this purpose from here.
SYBR® Green miRNA Detection
We recommend using the NCode™ VILO™ miRNA cDNA Synthesis Kit to generate cDNA. Multiple peaks in your melt curve could be from several sources:
- Contaminating gDNA—Make sure to DNase-treat your sample before the qPCR step.
- Primer concentration—We recommend a final concentration of ~200 nM for the miRNA-specific forward qPCR primer. Higher primer concentrations can lead to the generation of primer-dimers.
- Nonspecific products—Check the specificity of your primer to make sure it does not amplify at any other location.
- Primer-dimers—Primer-dimers can also form if there is no template present. Make sure to have at least 1 sample that can act as a positive control for your target.
The universal NCode™ primer is only provided in the NCode™ miRNA cDNA synthesis or NCode™ miRNA qRT-PCR kits, as enough volume is provided for the set number of reactions. The universal primer sequence is not provided.
Software Data Analysis
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 Ct values for U6 on the card, and see if it is in the expected range for your typical samples.
The TaqMan Advanced 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 some 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. Check the Ct values for your endogenous control assays on the card, and see if it is in the expected range for your typical samples.
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