Measuring gene expression

NewtoBio

wrote...

Posts: 9

Rep:

9 years ago

Hello I am really lost at the moment, can anyone explain to me the semi-quantitative RT-PCR process?

I don't understand how, by comparing a housekeeping genes band intestity (after PCR cycles and gel electrophoresis), we can measure the target gene expression?

What will the differences or similarities mean and say about the target gene?

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wizarddim

wrote...

9 years ago

This paper describes it well (attached).

In addition, this article has a good explanation: http://stanxterm.aecom.yu.edu/wiki/index.php?page=Semi_-_quantitative_RTPCR

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pmcm20.pdf (535.85 KB)

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NewtoBio Author

wrote...

9 years ago

Quote from: wizarddim (9 years ago)

This paper describes it well (attached).

In addition, this article has a good explanation: http://stanxterm.aecom.yu.edu/wiki/index.php?page=Semi_-_quantitative_RTPCR

Thanks so much, the only thing that is bothering me now is...

If I am using specific primers from my reference, and different specific primers for my target (in the same sample) what type of RT-PCR would this be? Competitive or another?

Thanks again

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bio_man

wrote...

Educator

9 years ago

Why don't you just use a regular PCR protocol?

Here are some sources that describe when to use competitive.

1) http://www.uni-ulm.de/~dkaufman/rt-pcr.html
2) http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/realtimepcr.htm
3) http://www.scielo.br/pdf/bjmbr/v36n12/4730.pdf
4) http://www.gene-quantification.com/competitive-rt-pcr.pdf

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4730.pdf (611.31 KB)

competitive-rt-pcr.pdf (214.92 KB)

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NewtoBio Author

wrote...

9 years ago

Quote from: bio_man (9 years ago)

Thanks for the links,
I was given a protocol; find specific primers for GADPH and specific primers for CD29 beta integrin.
Then theoretically conducted RT-PCR and ran them on a gel.

Because the primers are different, competitive PCR seems unlikely

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