RT-PCR

louis muff louismuff at hotmail.com
Wed Sep 19 06:15:45 EST 2001


I am a phd student who requires some assistance

We investigate the effect of cytokinins on the photosynthetic 
apparatus. To perform this, we work with transgenic plants which 
contain the ipt-gene coding for isopentenyltransferase. This enzyme 
catalyses the rate limiting step of the cytokinin biosynthesis. The 
gene is proceeded by a promoter which can be induced. We work with 
two types of transgenic plants, one construct with tetracycline 
induction and another with light induction. we want to compare the 
expression of different genes:

- the ipt-gene itself, to correlate induction of expression (by 
tetracycline / light) with the level of expression (mRNA) and the 
concentration cytokinins in the plants

- 2 chloroplast genes (to investigate the effect of cytokinins on the 
expression of these genes)

We would like to investigate the level of expression with RT-PCR. We 
designed primers for these genes ("Oligo"). We use the "i-cycler 
PCR-instrument of Biorad" to perform a quantitative RT-PCR so we need 
a intern standard (house-keeping gene) to compare with. We took the 
gene coding for ubiquitine. The primers we designed are not able to 
distinguish between RNA and DNA, it is therefore very important our 
RNA-samples are DNA-free. Both DNA (for PCR-experiments) and RNA are 
isolated with kits of Qiagen. However we have had a number of 
problems getting the pcr to work reproducibily, I would like some 
advice on the following questions:
- Do you perform quantitative RT-PCR on plant material and with a 
similar PCR-instrument? Do you have any experience with investigating 
expression of chloroplast coded genes?

- Do you use an intern standard and which one? Do you think 
ubiquitine is a good choice? It is coded by the nuclear genome, but 
we are also investigating the expression of some chloroplast genes. 
Do you think we can use the same intern standard or do we have to 
take an additional one coded by the chloroplast? If so, do you have 
any idea about which gene we can use?

I would be gratefull if anyone could help

please contact  louismuff at hotmail.com




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