Hello ,
I'm studying on genes involved in cancer, but lately I expose with some problems. These lately studied genes have very low expression levels and I can hardly detect their expression in tumor tissues as well as normal tissues.
I want to know which RNA extraction kit (from solid tumor tissue e.g. breast tissue) and cDNA synthesis kit are suitable for me or what do I do?
Thank you in advance.
Which endogenous control are you using. The problem might not be from the RNA extraction kit. However, we use the direct-zol RNA mini prep (RNA extraction) and the high capacity RT kit.
Thank you for answering my question,
B-actin and GAPDH as a internal control. I try RNA extraction using RNXplus, TRIZOL, miRNeasy Mini Kit, I'm getting very sharp bands for internal controls but no band for interested genes from RNXplus and TRIZOL and only very weak band combined with smear from miRNeasy Mini Kit. which high capacity RT kit you mean?
If everything is working, maybe they are just low expressing genes. You could try increasing RNA input, or a preamp kit if you are using qPCR for expression analysis.
I am using the applied biosystems high capacity reverse transcription kit. I will suggest you try using TBP as your internal control, I have used that for low expression genes.
Dear Bubaraye Robson Uko,
Thank you. Please can you (or other researchers) tell me other suitable cDNA synthesis kit for genes with very low expression?
i work with very low expression kit and so far the applied biosystems high capacity rt kit has worked perfectly for me. You can give it a try,
Dear Jennifer Gibbons,
Thank you.
I am using qPCR for expression analysis. Can I use preamp kit?! The results will be reliable? I mean, compared to internal control. Which preamp kit is suitable for me? or You usually use which one ?
As someone else said some genes are naturally low expressed. However, there are also genes whos mRNA are more degradable which might play a role in your material. I would not only look at which kits to use but also how the material is handled before RNA purification. Make sure that it has not been thawed and that the pieces are not to large so that is did not freeze at once. Also the way you lyse the tissue will have an effect on how much, I would recommend using a mechanical tissue lyser. Concerning kit we have good experience of Qiagens RNeasy lipid mini kit and Life Techs RT kit.
About endogenous controls, remember to use those that varies least in your co-hort.
Dear Pernilla Lång,
Thank you, yes I agree with you about degradable mRNA, and can I please ask you one more question? I am using qPCR for expression analysis. Can I use preamp kit?! The results will be reliable?
Theoretically it should be fine. We have not used ourselfs so I dont have personal experience from it.
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