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
