Hello scientists!
I'm working on gene expression stuff in rice plant, and I'm facing a little problem, which is that the starting quantity ,and expression of course,is so low on the qPCR n all samples!
the mass standards are so fine so that I exclude a primer dimer reason and the RNA concentration was so good from the beginning . so I'm suspecting now a problem with the cDNA protocol which may be not that good !
can you please provide me with good protocols that you find it better? I'd really appreciate any advise to deal with gene expression in rice plant particularly.
thank u already :)
Hello Hadeel,
Although I'm not working on plant gene expression, I have quite an experience with the expression studies of human genes. After the RNA isolation and quantification the very important step is to confirm the quality of the RNA by bleach gel electrophoresis. Assuming you have done that, which kit are you using for cDNA synthesis? I use Verso cDNA synthesis kit which never has failed for me till now. Here is the link for the details on the kit https://www.thermofisher.com/order/catalog/product/AB1453A
The kit insert has all the details you need to know.
Hope this helps.
Good day!
Hi Hadeel,
what is the concentration of RNA? If it is possible then to check quantity of RNA try to use Bioanalyzer.
To prepare cDNA, 60-100ng total RNA will yield accurate and precise results in qRT-PCR. Quality of RNA 260/280 ratio should >2.0 then you can get hopeful data.
For cDNA synthesis i will suggest you the SuperScript IV First-Strand Synthesis System for RT-PCR is optimized for synthesis of first-strand cDNA from purified poly(A) or from the total RNA. The kit is working well and the price is reasonable.
Good luck !
Hi,
Is your control genes such as GAPDH cycles are also coming very high? If yes then you have to increase the cDNA amount. We use Qiagen cDNA kit which can convert upto 500ng of RNA.
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