Almost from the moment the science recognized the value of PCR amplification, it has been a bit of a love-hate relationship. One of the latest additions to the PCR portfolio, real-time or quantitative PCR (qPCR), can be an amazingly powerful tool. However, just like traditional PCR, qPCR can be frustrating. There are a number of parameters that can influence the success of your qPCR assay. Below I have highlighted ten things to consider when trying to improve your qPCR results. Continue reading “Top 10 Things that Might Improve Your qPCR or RT-qPCR Assays”
I will admit that over the years, I have watched various crime scene investigation shows and read several books by Kathy Reichs and Patricia Cornwell because I was fascinated by forensic science. These same books and shows are a guilty pleasure because as a scientist, I know these portrayals do not accurately reflect how laboratory work is done. Answers are not so cut and dried as an exact estimation of time of death—for example, death was five hours before the body was found in an abandoned warehouse. However, scientists are always looking for ways to improve accuracy in time of death estimates, which are currently based on a few physical clues that are affected by environment and other factors. One approach taken by Sampaio-Silva et al. (1) was to assess the RNA degradation using reverse transcription quantitative PCR (RT-qPCR).
The authors of this PLOS ONE article wanted to determine if nucleic acid degradation could be used as a method to improve time of death estimates. Continue reading “A Molecular Approach to Estimating Time of Death”
Quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) are useful tools in laboratories around the world for absolute or relative quantification of DNA or RNA targets from biological samples. Once you understand the basic principles and have optimized the reaction and cycling parameters, these techniques tend to be rapid, accurate and easy-to-perform—makes me wish that qPCR and RT-qPCR were more commonplace when I worked in the lab and used radioactive Northern blots to quantitate my gene of interest. This is clearly another case of “If only I had known then what I know now”. While I’m no longer working in the lab, it’s not too late for you to benefit from a good introduction to qPCR and RT-qPCR, and on January 15, 2013, that’s exactly what one Promega Research and Development Scientist presented: A webinar entitled “Optimize Your qPCR and RT-qPCR Assays with Careful Planning and Design”.
Continue reading “Harnessing qPCR and RT-qPCR in Your Laboratory”
There are a lot of choices when it comes to reverse transcriptases. Choosing the correct one for your cDNA synthesis and RT-PCR project is important. Here are a few questions that will lead you to right RT for your application: Continue reading “Choosing the Right Reverse Transcriptase for Your Project”