Hello all,
what is your suggestion for choosing the best reference gene for qPCR in higher plants? My experiment is focused on different gene expression under various irradiance (only green light, only red light, etc.)
Thank you for ideas :)
Martin
Hi Martin,
I strongly recommend you to take a few RGs under consideration and select the best one for your experimental conditions. Standard reference genes are not always a good solution, especially 18S rRNA gene, which presents strong expression and give low Cq values, so you will probably have a big difference between the level of your GOIs expression and RG expression.
I usually start with RefGenes tool from Genevestigator and identify few genes with most stable expression, then add few of standard genes (usually GAPDH, UBI, TUB, ACT, and EF-1), prepare the validation and test the stability of expression in my samples with geNorm and NormFinder. There are a lot of papers dedicated to this issue.
The other thing is the number of RGs used in the experiment - you should use two or more RGs to convince the reviewers ;)
Good luck with your experiment.
Hello Martin,
For quantitative expression analysis in higher plants, you can select beta-actin, GAPDH, 18S RNA, protein phosphatase 2A, tubulin or ubiquitin as reference genes.
Best of Luck.
Hi Martin,
I strongly recommend you to take a few RGs under consideration and select the best one for your experimental conditions. Standard reference genes are not always a good solution, especially 18S rRNA gene, which presents strong expression and give low Cq values, so you will probably have a big difference between the level of your GOIs expression and RG expression.
I usually start with RefGenes tool from Genevestigator and identify few genes with most stable expression, then add few of standard genes (usually GAPDH, UBI, TUB, ACT, and EF-1), prepare the validation and test the stability of expression in my samples with geNorm and NormFinder. There are a lot of papers dedicated to this issue.
The other thing is the number of RGs used in the experiment - you should use two or more RGs to convince the reviewers ;)
Good luck with your experiment.
As mentioned above and seen by other researchers some reference genes would fluctuate under your conditions although they might not under standard growth conditions. That's why people use different references depend on their conditions. e.g. people in the circadian field use PP2A as a reference since the transcript doen not cycle. Please check published lit for your conditions and check few refs as Michal suggested.
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