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Increasing RNA yield from very small tissues


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#1 Breda3

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Posted 25 July 2014 - 07:54 AM

Hi,

 

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.

 

Breda



#2 bob1

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Posted 27 July 2014 - 12:55 AM

You might get a better yield from a tradition extraction (i.e. phenol/chlorform or trizol) with a carrier such as linear polyacrylamide.  This would especially be the case if you are used to doing these sorts of extractions.






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