I am working with very small kidney tissues from a fish(~0.5 mg or less) and I have been having difficulties getting consistently good yields of RNA from these tissues. I started off using QIAGEN's RNeasy minikit but was unable to obtain good yields so I switched to OMEGA's MicroElute Total RNA kit which improved my yields but did not remove gDNA contamination (I had positive NORT controls during qPCR). Therefore, I treated my RNA with Ambion's DNase Treatment Set but this really reduced by RNA yields so I tried an on-column treatment instead (Qiagen's DNase Treatment set). However, the on-column treatment appears to destroy my 260/230 ratios and I also have lower RNA yields than normal.
I have also tried different homogenization techniques to increase my yield including handheld homogenization, bead beating and pulse-sonication. I'd like to try grinding the tissue under liquid nitrogen with a mortar and pestle but the tissue is so small I fear I'll lose too much of it. The bead beating seems to work the best but it is still not great. I have reduced the input of RNA into my cDNA reaction which has produced successful results in qPCR but some of my tissues produce yield as low as 5 ng/uL which is lower than I'd like.
Does anyone have any suggestions? Pooling the samples is not an option due to limited sample size.