I need to amplify a gene whose expression is very low in almost any cDNA I get. I want to know where or at what stage of a plant this gene is expressed the most. According the RNA sequencing analysis report of our lab, this gene is supposed to be highly expressed in Senescent flower petals, but actually it is not. With that cDNA in hand, its expression is somewhat visible while using high fidelity polymerase, but that's too expensive and our lab can't afford it much. So I only got the common Taq mix to do the amplification. Is there any online webtool in NCBI etc for this purpose???
Please help me with it,,,
Thanks
You should do a Northern analysis of the gene with different parts of a plant-of-interest in different development stage (some gene only expresses at a specific plant development stage).
Guowang Liao Thank you. I can only do qRT-PCR if I get a good quality cDNA with highest expression of that gene, with highly specific primers set. I don't want full length, but only the CDS...
Yuan-Yeu Ya Thank you. With that procedure, it will be nearly hundreds of samples, which is too in-adequate. And BTW we know about northern blot, how tricky it is...
Actually I found one method in Arabidopsis TAIR database, which mentions full map of the genes expression in different parts of the plant. But thats only for Arabidopsis, I hope there is some for other species too
Firstly isolate RNA -> make cDNA ->do real time PCR to understand the expression level of the gene.- I hope you have done this.
2ndly- if possible understand the pathway in which the gene product is involved, then read about the pathway, in which organ or tissue the pathway is most active, then you can investigate on that organ or tissue.
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