I need to assess the gene expression of a newly identified gene of a fungus, and also I need to see the effect of environmental conditions such as Temperature, pH for the gene expression.
Do not forget that "gene expression" does not mean mRNA level but protein level!!!!
Sometimes you have high level of mRNA with very bad translation efficiency and small level of proteins. And even for one specific mRNA the translation efficiency might vary under environmental conditions including pH modulation.
there are methods to quantify the expression in environmental conditions. You could use two different methods First one is simplest one you can amplify your desired gene with Sybr Green Dye and specific primers and quantify the expression.
second make a specific probe for gene along with primers and use them for quantification with TaqMan chemistry method. This method is more stringent and precise.
you need a positive standard also for Syber Green method with known number of copies.
So you apply to different conditions to your fungus, and also prepare control, after that just isolate RNA (immediately after stopping the treatment) from all your experiment combinations. Next step is to do reverse transcription reaction to get cDNA for the samples and run qPCR reaction. Remember to prepare all samples in at least three biological conditions, and for all samples do 2 technical repeats. You need also same housekeeping gene (the expression of this gene is constant no matter what kind of conditions you use), which is base for calculation of expression of gene of interest. How to calculate everything you find in here http://www.ncbi.nlm.nih.gov/pubmed/18546601. Good luck
Hi. you need to find a control gene which was used as control in other studies. you use control gene to assess changes of your interested gene with it in other hand it is your ruler :). If the control gene did not determined in your organism, you must identify it. After determination of control gene you can apply your treatment and start sampling. after that extract RNA from your samples and convert them to cDNA and then do the real-time for measurement of changes in your interested gene.
use article which proposed by Magdalena Stolarek at above to training and to perform calculations .
I hope that is helpful
Since you are going to be measuring this under different conditions, I would suggest you pick several potential housekeeping genes (that is, perhaps as many as 5 or 6). The reason being if you use only one, and it does not behave as expected under a particular experimental condition, then you have no housekeeping gene for a baseline. If you select several, odds are better that at least one will be suitable for any given experiment.
Hi Ruvishika.... as you have mentioned you want to check 1) the expression of your newly identified gene as well as 2) expression of that gene under various environmental conditions. For the first objective you just need to compare your new gene expression with the housekeeping genes ( you can use number of housekeeping genes but minimum two is required). As already mentioned by other members for that you need to extract RNA, then prepare cDNA and then run your real-time PCR experiment for both the new gene as well as housekeeping genes at the same time. Then after comparing the ct values of your gene of interest with housekeeping genes, you can calculate the expression of your gene.
Similarly, you can study the expression of your gene under different environmental set ups. But in this case you also need a control condition with whom you will compare the environmental set ups.
I have done similar work in my PhD thesis, if you need further information, don't hesitate to ask me.
In addition to what others have suggested, you must pay special attention to sample collection. If you are assessing different environments, it is important to collect and freeze the samples as quickly as possible (i.e. put in liquid nitrogen while you get your samples to the lab, store at -80 C and freeze-dry if longer storage needed. Then use several internal controls as others have mentioned above.
At the first you must select a suitable housekeeping gene (control gene) for your samples. you can identify it with studies. Then you must do a RT-PCR with SYBR green (with melting curve analysis) or appropriate probe and at the end compare Ct values of your desired gene with housekeeping Ct values. Dont forget a control condition to compare with various enviromental conditions and 3 biological and 2 technical repeats.
The stages you must follow are: 1- sampling 2- RNA extraction 3- concentration and quality identification of RNAs 3- DNase treatment 4- cDNA synthesis 5- primer designing for your desired gene and of course housekeeping gene determination 6- RT-PCR 7- data analysis.
Good luck.
Do not forget that "gene expression" does not mean mRNA level but protein level!!!!
Sometimes you have high level of mRNA with very bad translation efficiency and small level of proteins. And even for one specific mRNA the translation efficiency might vary under environmental conditions including pH modulation.
I used design expert software to design my experiment with temperature and pH as the parameters and gene expression as a response but you must identify first which gene you are really want to observe. optimize the real time PCR conditions first.
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