Hi all,
In my lab we are designing some acute osmotic and salt treatments in plants of a endemic tomato variety to analyze the relative transcript levels of different genes by qRT-PCR at different times. One of the discussion we are having is how to perform the sampling. In one hand, some believe that the best is to pool samples and then perform the RNA extraction (3 plant per pool and 2 pool) and in other hand some believe in perform the RNA extraction and qRT-PCR experiments in each individual without pooling samples.
What do you recommend is the best approach?
Thanks!
Hi Luis,
We usually pool the samples of 5 plants at each treatment for RNA extraction. Equal weight is essential for reliable results. Extracting by individuals is more costy, and you can bring mutch inaccuracy into the experiment. I hope I could help!
I believe that qRT-PCR of the genes of each individual wouldn't give you many answers. As already stated it will bring a lot of variation in the experiment. It would be more sound to do some pooling and make sure you've got at least 3 biological replicates for each of your treatments. For example you could pool x individuals together into one pool and do that for at least 3 pools. I usually do qRT-PCR on individual plants if THAT plant has a special phenotype that others might not have but I'm very careful in interpreting these results.
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