I want to quantify RNA plant viruses using RT-qPCR. I have some primers that I found in a publication. The primers were used witha taqman probe. However, I have SYBR green mastermix in the lab. Is it OK to use these primers? Will they work?
Hi,
Yes you can. We found the primers for EMCV in the publications too. It was designed for TaqMan, but in our research we use with SYBR green mastermix. And we have well results.
Yes they can, so long as you check on a few things, like the location of the probe, the target site and organism should be exactly the same as the one in the paper. See the two links below, they answer your question.
http://www.protocol-online.org/biology-forums-2/posts/6337.html
https://www.researchgate.net/post/Using_primers_in_Taqman_and_SYBR_green_techniques
Thank you people for the feedback. I will try and keep you posted on my results.
Dye-based assays are perfect for many real-time qPCR applications. When you understand the true advantages and disadvantages of probe-based assays, you can reduce the cost while keeping the quality of your qPCR experiments high.
Novice qPCR users often argue that data generated with dye-based assays are susceptible to misinterpretation: the detection of nonspecific PCR products would influence the Cq values.
But the detection of nonspecific PCR products should not be cancelled out by adding a probe to the qPCR reaction. It is through careful experiment design and optimization that you obtain specific, efficient and sensitive amplification and detection of your target of interest. Primer concentrations, denaturation and annealing times and temperatures, and buffer composition are few of many critical parameters that contribute to a robust qPCR reaction. When sensitivity is not an aspect that you want to improve, optimizing these parameters will give you a suitable qPCR assay. And it does not need a probe. This is the strategy applied by Biogazelle when designing Bio-Rad's PrimePCR assays: the most comprehensive portfolio of validated Research Use Only dye-based qPCR assays for human, mouse and rat.
You can find additional arguments, and additional advice on when to use what detection technology at https://blog.qbaseplus.com/taqman-probe-or-sybr-green-dye
- Badges
- Science method