Posts Tagged ‘qPCR’

Video Introduction to Droplet Digital PCR

 :: Posted by American Biotechnologist on 12-05-2011

As a follow up to Bio-Rad’s acquisition of QuantaLife, we are proud to present the following educational training video: “An Introduction to Droplet Digital PCR”, given by award winning field application specialist Dr. Sean Taylor.

Be sure to stay tuned for the advanced training video which will be posted in the near future.

qPCR delta-delta Ct method simplified

 :: Posted by American Biotechnologist on 11-01-2011

I found this nice, down to earth explanation of the delta-delta Ct method for calculating qPCR results (i.e. qPCR results corrected with internal controls).

A Practical Approach to Assay Design for qPCR

 :: Posted by American Biotechnologist on 09-02-2011

Designing good qPCR assays can be fun! Learn how to overcome difficult assays, designs and optimization while conforming to the MIQE guidelines.

Increase the amount of data from your RNA samples

 :: Posted by American Biotechnologist on 08-24-2011

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.

Bio-Rad Laboratories has recently launched the iScript advanced cDNA sythesis kit for RT-qPCR as an answer to some of these challenges.

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

How many reference genes do you use in qPCR?

 :: Posted by American Biotechnologist on 08-10-2011

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!

How many reference genes are you using in your qPCR experiment?

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