Can Ct's greater than a cut-off be considered valid results?
Yes they can. However, it is important to recognize the true linearity and detection limits of your assay: Ct values above the cut-off can indicate non-specific amplification, unless your NTC is a true- no-target control, and you have run a statistically significant number of replicates. Any results with Ct above the recommended cut-off need to be validated with individual assays on plates.
What is a good Ct cut-off for the TaqMan MicroRNA Array Cards and TaqMan Advanced miRNA Array Cards? In other words, beyond what Ct should I not trust the data?
The typical Ct cut-off on TaqMan Array Cards is 32, which is equivalent to Ct 35 on a plate (10 µl reaction). Previous studies show that if you use pre-amplification, a Ct cut-off of 29 or 30 can be used to reduce numbers of false positives (see Technical Note Optimized protocols for human or rodent microRNA profiling with precious samples). To ensure that you have selected a correct cut-off, you should run replicates of the same sample and use Ct cut-off before you see an increase in the Standard Deviation.
I want to purchase TaqMan Array Human MicroRNA A Cards v2.0 (Cat. No. 4398965). What other reagents do I need to purchase in addition to these cards?
In order to perform reverse transcription on these cards, you will need the following:
- TaqMan MicroRNA Reverse Transcription Kit (Cat. No. 4366596)
- Megaplex RT Primers pools (for human Type A cards Cat. No. 4399966, for Type B cards Cat. No. 4444281)
You can proceed with or without a preamplification step which depends on the total RNA amount. Total RNA amount of 1‐1000 ng supports reverse transcription reaction with preamplification, whereas 350‐1000 ng of total RNA supports a reaction without preamplification.
In case you are performing preamplification you would also need the TaqMan PreAmp Master Mix (Cat. No. 4391128) and the Megaplex PreAmp Primers, Human Pool A v2.1 (Cat. No. 4399233).
You will also need the Master Mix for the qPCR reaction. It is not included in the card kit. The recommended Master Mix is: TaqMan Fast Advanced Master Mix – run mode Fast (Cat. No. 4444556)
For further information, please see the following link.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
Why does the negative control well show amplification when doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards?
Most likely the reagents or the cDNA template are contaminated. Please follow established PCR laboratory best practices.
Why do I have poor reproducibility across technical replicates when doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards?
Most likely the reagents were not adequately mixed. Ensure that all samples and reagents are mixed well.
