I am trying to extract good quality RNA from 6mm ear punches from CD-1 mice. I use sterile biopsy punches, place the individual punches in cryo vials and then immediately into liq. nit. They go into -80 until processing. I use a Qiazol and Qiagen RNeasy Mini Kit to extract RNA. My Nanodrop values are great, good 260/230 and 260/280 values, but running RNA on gels show smears, confirming that RNA has degraded. I have spent 1 day going from extraction to the gel so RNA has not been frozen and thawed multiple times.
Any suggestions? Is it that I need a special extraction kit for ear/cartilage/skin?
- You have not attached any image of your gel so its hard to troubleshoot
- What is the purpose of RNA extraction?
- Generally, RNA does not give only strong bands without any smear. And that smear is not just because of the degraded RNA, but there are several types of RNA, which are of different sizes and these results in the smear
- Normally, if RNA is needed to be used for the qPCR analysis, cDNA is made. RNA is mostly not used for gel electrophoresis. However, if gel electrophoresis is needed, the protocol of gel prep and buffer is not as same as DNA gel electrophoresis. So, please get correct protocol and follow it
- If RNA is needed for some special analysis like RNA-seq, the qualitative analysis need to be done by bioanalyzer, which gives much detailed information of the quality of RNA
Abhijeet Singh
- I attached two images. The first is what seems to be bands on some RNA samples I ran, the other image shows no bands on other RNA samples. All are RNA obtained from ~7mg of ear punches.
-The purpose of the RNA extraction is for qPCR analysis. Previously, I would get 'no amplification' errors after completing my qPCR on my cDNA samples. I figured that was because the RNA was degraded to begin with (I know it probably can't be anything else because I made sure my kits and primers were okay by testing other people's cDNA).
-I used RNA gel electrophoresis to try to confirm the integrity of my RNA, whether it was degraded or not.
-In general, the end goal is for me to test inflammatory markers. I have yet to get proper amplification of any of my cDNA. I assumed the problem was with the RNA extraction of the ear punches.
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