I have sprayed my 30d old plants with Gibberelllic acid (GA3)(100ppm) and collected sample for RNA isolation after 2h,4h,8h,12h and 24h of spray. After that I did q-Rt for GA biosynthetic genes with this RNA samples to know how this gene expression changes with the spray of GA3. so , I was expecting that , say for GA20 oxidase, expression will low in 2h and after a certain period it will start up- regulate and will be equal to control i mean sample without GA spray. But I am getting result like, it down regulate at 2 h, up regulate at 8h and again down regulate at 24 h. So, i cant come to a conclusion. Can any one please give me suggestion , why i am getting such a abnormal expression change? Thanks in advance.
The first thing that could explain your result is your reference gene, you are not mentionning it, what is it ? Actin, GAPDH ? If your reference gene is modulated by your treatment all your final values will be shewed, in all QPCR experiment the first step is to ensure that the reference genes that you are using are not affected by your treatment. Have you checked that ?
I think whatever you are getting is right if you do not have any technical problem.... watch your reference gene first. Try to optimize the qPCR with atlas three reference gene. Only GA treatment is not enough watch out other surrounding conditions too.
Also something to consider are the samples you harvested. I assume you took whole leaves (which plant?) Make sure you take leaves from the very same developmental stage. Are your GA genes somehow coupled with circadian clock, this might also be a thing to consider. Good luck
Check your reference gene .Same reference gene in Arabidopsis can show a different pattern than in Jute when sprayed with GA. check other experiments with GA in other crops for a good reference gene
Thanks to all. I have used protein phosphatase2 as my reference gene. I am not sure whether it is coupled with circadian clock, checking it. If so what should I need to do?
- Badges
- Science method