Designing good qPCR assays can be fun! Learn how to overcome difficult assays, designs and optimization while conforming to the MIQE guidelines.
Archive for the ‘qPCR tutorial’ Category
In the past, we’ve discussed the importance of selecting appropriate reference genes for your qPCR experiment (also see point 7 of the MIQE guideline checklist). This means that it is important to select genes that do NOT exhibit any changes in expression under the treatment conditions you are studying. This is easier said than done!
“Once upon a time” everyone used either beta actin, 18s, or gapdh as reference genes. Their expression never changes, right? Wrong! So which genes should you choose? If you try to figure it out using previous papers, how do you know that they’ve chosen the correct genes? If you run a few genes side-by-side and try to compare their expression both under treatment and control, which one should you set as the baseline and which one can you say is for sure moving (it’s all relative isn’t it)?
One of my twitter friends told me that she uses six reference genes in her qPCR experiments. I used to use two. That got me thinking…how many reference genes does the “average” lab use? Please help satisfy my curiosity by participating in the poll below!
In honor of this launch, we invite you to review some of the resources (including technical notes, review articles and video tutorials) that we have posted on high resolution melt analysis. Feel free to to click on any of the links below for further details:
- High Resolution Melt Analysis Applicatons (publication)
- The Versatility of High-Resolution Melt Analysis (2 resources: review article and technical note)
- Educational Webinar: High-Resolution Melt Analysis (archived version available, registration required)
- A Practical Guide to High Resolution Melt Analysis Genotyping (technical note)
- A Video Tutorial for High Resolution Melt Analysis
- PCR Assay for Chromatin Accessibility (epigenetics methods article)
We have written many posts about the MIQE real time PCR standards that are basic requirements for anyone engaged in real time PCR experimets. Now there is a new tool for all ipod/iphone users to add to their arsenal. A MIQE qPCR app!
The MIQE app helps scientitst review scientific work and check their own project’s MIQE compliance. Plus, the app includes a list of the most current qPCR news and events and “emergency” contact numbers that you can call/email should you have any questions about your qPCR experiments.
Here’s a a great powerpoint presentation by Frank Bizouarn, International Field Application Specialist, Bio-Rad Laboratories, on how to perform fast qPCR for high throughput screening.
The presentation goes through the intricacies of primer design, ideal amplicon characteristics, troubleshooting with a thermal gradient, how reagents can impact your experiment, primer titration, assay sensitivity, standard curves, the affect of PCR inhibitors, how scale affects reproducibility, throughput and speed.