Designing good qPCR assays can be fun! Learn how to overcome difficult assays, designs and optimization while conforming to the MIQE guidelines.
Posts Tagged ‘qPCR’
Some of the technical challenges in qPCR research can be traced back to the reverse transcription step and the synthesis of cDNA for qPCR analysis.
Limitations in the maximum amount of template RNA that can be added to an RT reaction make it tougher to detect low-abundant target genes and limits the amount of data that can be obtained from a typical 20ul RT reaction.
iScript advanced is a 2-tube format cDNA synthesis kit with a short protocol (35 minutes) and an allowed RNA input amount of up to 7.5ug.
The kit will:
- increase the amount of data that can be obtained from an RT reaction (great for high-throughput labs)
- enable you to analyze a larger number of target genes from a single sample
- increase the sensitivity for certain target genes due to higher RNA input capacity
- cover a broad dyamic range and sensitivity
For more information on iScript Advanced including experimental data download the iScript Advanced product bulletin.
To read more about Bio-Rad’s qPCR solutions visit www.bio-rad.com/qpcr
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)
In this slideshow, you will learn the latest epigenetic techniques including:
- discriminating epigenetically inactive chromatin from active chromatin
- discriminating between aberrant and Monoallelic DNA methylation
- predicting gene expression levels via chromatin structure assay
- analyzing how DNA methylation affects promoter activity