Fast qPCR assay optimization and validation techniques for HTS

Following hot on the heals of yesterdays post “A Practical Approach to Assay Design for qPCR“, we are proud to present you with another practical SlideShare on Fast qPCR assay optimization and validation techniques for HTS (high throughput screening). As with the previous presentation, the slide deck can be maximized for easier reading.

Related posts:
How to ensure significant results from your qPCR studies
Applications of MIQE to Real Time Quantitative PCR
A Practical Guide to High Resolution Melt Analysis Genotyping

A Practical Approach to Assay Design for qPCR

Designing good qPCR assays can be fun! Have a look at the presentation below to learn how to overcome difficult assays, designs and optimization while conforming to MIQE guidelines. If the slides are hard to read in their current format, click on the full screen button on the bottom right corner of the slide deck to enlarge.

How to ensure significant results from your qPCR studies

So you’ve heard all the hoopla about MIQE and how important it is to follow the guidelines when conducting your real time qPCR experiments, (if you haven’t, you better check this out!), but where’s the proof that following MIQE actually makes a difference? After all, qPCR was around for several years before anyone came up with this MIQE stuff. Right? Well…maybe not! As it turns out, qPCR experiments that don’t follow MIQE guidelines can be very difficult for others to reproduce and can even lead to incorrect conclusions in gene expression studies.

In a recently released case study involving breast cancer patients, researchers found that the MIQE guidelines played a central role in obtaining the expected conclusions with a positive control target. The article was written to show readers in a simple, stepwise process how to design a good qPCR experiment that covers the major components of the MIQE guidelines. While each step of the experimental design was found to impact the final conclusion (sample collection, RNA quality and purity and the use of appropriate primers), the most striking result was the impact of reference gene selection on the results. At one extreme, normalization by the commonly used GAPDH and 18S reference genes gave either no significant results of statistically significant data that was opposite to the expected outcome, while other more stable reference genes, (HPRT1 and TBP), gave statistically significant data that supported the conclusions from previously published results with this target.

The study concludes that the application of the MIQE guidelines to qPCR experiments result in reliable, quantifiable and reproducible data. With a growing list of journals that are now requiring the submission of supplemental data supporting adherence to the MIQE guidelines,the publication of qPCR data will become more challenging if they are ignored.

So don’t miss out on significant data. Use MIQE!

To read the case study click here.

PCR: Rescuing the reputation of Canadian salmon

There has been much ado lately threatening the reputation of British Columbia’s fishing industry. According to an October 28th New York Times report, the deadly Infectious Salmon Anemia (ISA) virus was detected in Pacific salmon in BC which, if true, could have had a “deep impact on the survival of salmon in the Pacific Northwest.” As a point of reference, in 2008, the wholesale value of wild salmon sold by BC fisheries totalled approximately $135.2 Million. Alas, the Canadian Food Inspection Agency (CFIA) has just confirmed that the NYT report was indeed much ado about nothing.

Are you using the right reference genes?

Considering Real-Time PCR for gene expression analysis? Have you tested multiple reference genes or are you just going to run with your favorite such as 18S? Check out this article on suitable reference gene selection before you move forward. It could save you lots of grief in the long run.

Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses
Li-Yun Fu et al. BMC Cancer 2009, 9:49

Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different clinical outcomes.

Full Reference:
Fu, L., Jia, H., Dong, Q., Wu, J., Zhao, Y., Zhou, H., Ren, N., Ye, Q., & Qin, L. (2009). Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses BMC Cancer, 9 (1) DOI: 10.1186/1471-2407-9-49