Title: Measuring gene expression Post by: NewtoBio on Dec 1, 2014 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? Title: Re: Measuring gene expression Post by: wizarddim on Dec 1, 2014 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 Title: Re: Measuring gene expression Post by: NewtoBio on Dec 1, 2014 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 Title: Re: Measuring gene expression Post by: bio_man on Dec 1, 2014 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 Title: Re: Measuring gene expression Post by: NewtoBio on Dec 1, 2014 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 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 |