Dear Researcher,
First of all, thanks for reading my post. As i am working with a Arabidopsis knockout mutant that showed several phenotypes; one of it showed higher survival rate after heat treatment, What i was doing is to stratified the seeds on MS media for 3 days and let it grow in 23LD for 7 days and then do heating at 45degree for around 1 hour. As compared to WT, my mutant lines showed higher survival rates after 3days recovery. As the mutant i am studying is involved in pre-mRNA splicing and spliceosome assembly, i would like to check the alternative splicing of those heat shock genes through RT-PCR to see if there is any different in that, My question is it valid if i also do the exact procedure and then heating it at 45 degree for 1hour and then i straightaway do the RNA sampling. Do i need the plant to recover first or i can sample it right after 1hour? and what could be heat shock gene that i can test of including HsfA2, Hsp101? thank you!!
Hi Ke Chien,
your results sound interesting. The heat treatment you used most likely determines the basal thermotolerance of the plants (please refer to the definition of thermotolerance in our review: Yeh et al. 2012, Plant Science 195:10). However, usually Arabidopsis 7-d old seedlings do not survive at 44 degree for more than 30 minutes if heating is done in water bath. I guess you have used oven or incubator for the heat treatment, right? If so, you are likely measuring the acquired thermotolerance. I don't quite understand why you want to check the alternative splicing of HSP genes specifically? But I know that HSFA2 mRNA has been reported to be alternatively spliced (see Liu et al. 2013 Plant Physiol 162: 512).
Hi Ke Chien, It would be interesting to look at the response of your mutant to heat treatment at cellular lever. Our lab developed a simple assay (Hogg et al., 2011) that allows determination of programmed cell death rates induced by different treatments (including heat) based on observation of dying root hair morphology. Could be interesting to subject your mutant and its wild type to cell death inducing heat treatment (e.g. 49C for 10 min) and then determine the rates of programmed cell death in their root hairs after 5-6 hours. About the RNA sampling, I would do preliminary experiment with different time points first and then decide which is most interesting.
Hi Ke Chien,
As suggested by Joanna kacprzyk, I would also do preliminary experiment with different time points first and then decide which is most interesting. Regarding sampling, I would suggest to go for sampling straightaway after exposing it to heat treatments for one hour or whatever is more interesting for you. I do not think, it is a good idea to let them recover first, if you are interested to see expression level of heat shock genes.
Hi!
I would say that the sampling moment mostly depends on when your protein of interest is expressed and produced: if it is induced by heat, for example, it would be sensible to wait a few hours after heat shock to allow it to be produced. If however it is always present, then you can just do 30 minutes of heat stress. Indeed, most of HSPs are induced in just few minutes; besides, the more you wait, the less transcript you'll keep of those genes, as they stop being transcribed when the stress is over. So, depending on the expression profile of your gene of interest, I would sample either after 30 minutes of a few hours, but I wouldn't wait for days.
For genes to look at, just look at the TAIR website which HS related genes are predicted to have several splicing variants, even if not yet verified.
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