Megaplex™ RT Primers, Human Pool B v3.0 - FAQs

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)

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.

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards, why are the Ct values for the endogenous controls highly variable across the sample set?

Highly variable values for endogenous controls is most likely due to biological variation. We recommend that you use an alternative endogenous control, such as a non-variable miRNA.

Why is the Ct value for the no-template control (NTC) <35 for some TaqMan microRNA Assays?

The Ct value for the NTC can be <35 for the following reasons:
- There are non-specific interactions between primers. For NTC information on a specific assay, see the Megaplex Assay Performance File.
- The cDNA template is contaminated. Please ollow established PCR good laboratory practices to prevent contamination.
- The preamplification product was not diluted properly before real-time PCR. Ensure that you dilute the preamplification product according the procedure in the user guide.

Why can I not detect any miRNA in my sample using the TaqMan MicroRNA Assay?

Most likely, the threshold is set too high to detect miRNA in samples with low levels of expression. We recommend adjusting the threshold (Ct) or NOAMP flag threshold (Crt) to an appropriate level.

Why is the baseline variable when doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards?

Most likely the dried-down assays on the card were reconstituted at different rates, causing a dip in the early cycles of the baseline. We recommend that you use the relative threshold algorithm (Crt), which can correct for a variable baseline. If the software specific to your instrument does not have the relative threshold algorithm use the Relative Quantification app on Thermo Fisher Connect.

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards, why do my technical replicates have poor precision (standard deviation >0.5 for assays with a Ct value <30)?

Please review the following possible causes and solutions:
- Bubbles in wells. Use proper pipetting techniques to avoid introducing air into the fill reservoirs. Consider omitting the leaking wells from analysis.
- Wells leaking due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. We recommend that you consider omitting the leaking wells from analysis.
IMPORTANT: Do not move the carriage back across the card.
- Not all of the positions in the card holder were filled before centrifuging. Ensure that all of the empty positions of the card holder are filled with blank balance cards before centrifuging.
- The cards were centrifuged using a non-verified centrifuge. We recommend that you use a Sorvall centrifuge to prepare cards. Please see the Resources section at thermofisher.com/taqmanarrays for a list of compatible centrifuges.
- The dried-down assays on the card were reconstituted at different rates, causing a dip in the early cycles of the baseline. We recommend that you analyze results with the relative threshold algorithm (Crt) instead of the baseline threshold algorithm (Ct).

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Card, why is there no amplification within or across one or more rows of the card?

Please review the following possible causes and solutions:
- Empty wells due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. Do not move the carriage back across the card.
- Empty wells due to misalignment of the TaqMan Array Card Sealer. If the TaqMan Array Card Sealer is misaligned, contact Support.
-PCR reaction mix improperly prepared. Ensure that all reaction components were added to the PCR reaction mix.

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Card, why is there no amplification in some wells?

Usually, empty wells are due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. Do not move the carriage back across the card.

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Card, why is there no amplification for portions of the card?

Most likely the card was misaligned in the block during the instrument run. Inspect the card for crushed or distorted feet. If there are damaged feet, contact Support.

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards, why is there noise in the amplification plots for portions of the card?

Most likely the card was misaligned in the block during the instrument run. Inspect the card for crushed or distorted feet. If there are damaged feet, contact Support.

When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards, why is there gradient signal across the card?

This gradient signal indicates that the card was in a diagonal position during centrifugation because not all of the positions in the card holder were filled. We recommend that you repeat the assay with a new card and ensure that all the positions in the centrifuge card holder are filled.