Important Notice: Our web hosting provider recently started charging us for additional visits, which was unexpected. In response, we're seeking donations. Depending on the situation, we may explore different monetization options for our Community and Expert Contributors. It's crucial to provide more returns for their expertise and offer more Expert Validated Answers or AI Validated Answers. Learn more about our hosting issue here.

Why are my qPCR Ct values too high (> 35 or not detectable) in my qRT-PCR assay?

0
Posted

Why are my qPCR Ct values too high (> 35 or not detectable) in my qRT-PCR assay?

0

There are several reasons for not seeing a PCR product. 1. The corresponding gene may not be expressed above the limit of detection of the qRT-PCR assay method. 2. There may have been experimental error, in which case, use a template known to contain the gene of interest as a positive control to troubleshoot the PCR reagents and experimental procedure. 3. The RNA may have been of poor quality, in which case, be sure to perform all of the recommended quality control checks on the RNA sample (see Sample Preparation FAQs, above). 4. There may not have been enough template, in which case, use more input total RNA, or use the template at a lower dilution factor (higher concentration), or use a larger volume of template. 5. Another possible explanation pertains to when one is trying to detect cellular expression from an exogenous vector that has been introduced into a cell. If the vector expresses only the open reading frame (ORF) of the gene of interest, and the qPCR primers being used ampl

Related Questions

Thanksgiving questions

*Sadly, we had to bring back ads too. Hopefully more targeted.