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General troubleshooting for low product/late Ct values:

If you are observing unusually late Ct values, try diluting the template RNA. By doing this, you are diluting any inhibitors which may be present to a concentration where they do not inhibit the reaction. Additionally, try increasing the reverse transcription step to 55 °C and increasing the annealing/extension temperatures. This may help resolve difficulties caused by secondary structures present in the RNA template and/or primers.

In cases where reaction inhibition may be involved, try reducing the amount of template1 or add 0.4 – 4.4 mg/ml BSA to the reaction2.

For more specific problems contact [email protected] with the following information:

Amplicon size

Reaction setup

Cycling conditions

Screen grabs of amplification traces and melting profile


1  Scipioni et al. A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition. Virology Journal.5:94 (2008). doi: 10.1186/1743-422X-5-94

2  Plante et al. The use of bovine serum albumin to improve the RT‐qPCR detection of foodborne viruses rinsed from vegetable surfaces. Applied Microbiology. 52:3 (2010) doi: https://doi.org/10.1111/j.1472-765X.2010.02989.x


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