I'm using the Qiagen RNeasy Fibrous Tissue Mini Kit to isolate RNA from skin. Recently, my RNA quality (RIN) has gone down, and I'm trying to figure out why. One change I made in my protocol was switching from eluting RNA with 50ul of RNase-free water, and running the eluate through the column a second time, to eluting twice with 30ul of RNase-free water (the kit says to elute with 30-50ul of RNase-free water). Could this make any difference in quality (regardless of effects on concentration/yield)? The only other changes I made were additional steps to reduce salt carry-over (rolling the spin column with wash buffer, doing the optional extra spin) and I don't see how these could impact the RIN.
I use RNeasy kits too (but for yeast RNA, same principle).
Volume of eluting solution wont affect quality. Generally, if you use a smaller volume but put it through the column twice, you should get a more concentrated yield but quality shouldn't suffer. I elute twice with RNase free water and normally get great A260/280 and A260/230 ratios, so that shouldn't be a problem either.
Depending on the ratios you're getting (which dictates the source of contamination), it could be ineffective DNase treatment, ineffective protein precipitation/washing so protein carryover, or contaminated RNase free water..
You elute your RNA using 20ul of RNase free water. In other steps, after addition of each buffer you wait for 2-3minutes.Do not centrifuge immediately. Water should be RNase free
I have been using QIAGEN kits for RNA extraction from tissues and used 50ul of RNase free water provided with kit for elution. According to my experience I don't think that reaction volume or eluting twice effect the RIN value (i.e quality). But make sure that handling of tissue and all the processing steps are done as recommended and with precaution. I have also observed that the quality of B-mercaptoethanol used in RLT buffer does effect RIN, preferably use mercaptoetanol stored at 4 degree.
I have used qiagen RNA easy plant mini kit for 3 years. The main step that cause problem for RNA isolation with Qiagen kits is Alcohol step. What I mean is this, while you are pipeting your extract with ethanol kindly. You should be complately sure that any of compact particule or any of visuable particule in your mixture. Because of the fact that, if you do not mix your extract with etanol perfectly the mixture will choke the filter system. Please be sure for this step.
Eluting twice does not affect the RNA quality in my experience. Check water for contamination and try eluting with fresh RNase free water. Also, check A230, A260, A280 ratios for indication of contamination.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA