I m isolating RNA from wound tissue (rat) with good quality of 260/230 ratio however it gives no expression for collagen I, GAPDH neither VEGF in RT-PCR. RT-PCR was working fine with rna samples from heart of the rat. Is it possible that primers are not working for skin tissue? or smthng wrong with RNA isolation?
Because the conditions of the starting material you may have some inhibitors for both RT reaction and the PCR in your samples. If you dilute the samples 50 or even 100 times, may work, the technique is very sensitive in term of detection. If not, may be the quality (260/230) is good but too fragmented.
Skin collagen may be preventing access of lysis buffers into the tissues and thus you are getting lower RNA yields or higher protein contamination. As mentioned above 260, 280 ratios and denaturing gels would help gauge the RNA quality. Depends, if you are using manual methods or kits to obtain RNA. Enrichment of mRNA by Qiagen kits would also help !
It's already an advantage for you, as you are extracting the RNA from animal tissue, and therefore you can recover a high amount of good quality RNA with not too much tissue. So maybe you can try to use less tissue and not too much lysis reagent. This will help you to not be to hard when homogenizing the mixture, as RNA can be too fragmented and damaged at the end. By doing so the quality of the RNA will be preserved. And also do not leave the homogenate in the presence of the lysis reagent too long, especially at room temperature. Hope that this will help.
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