RNA extractions

I started the RNA extractions yesterday. I am using an Omega EZNA miRNA Kit and including the "optional" on-column DNase treatment. I'm planning to sequence almost every molecule in these so I want to make sure that I don't have any DNA contamination. The first sample I did was a throw-away sample to work through any bugs. Which was good cause there were a couple.

The first main problem I had was after I ground the tissue on liquid Nitrogen, I had to measure out 30-50mg, and the sample thawed. In the end my RNA Integrity Number (RIN) off the bioanalyzer was 7.8 (the higher the better - I'm shooting for 10, we may consider sequencing a sample that is as low as 9). I am pretty sure that the brief thaw was the issue there.

The second problem I had was that it calls for a 10 minutes spin at 4c and I don't have a centrifuge that refrigerates. I did the spin at RT and later found out that the lab next door has a centrifuge that will keep  it at 4c. This may have also been part of the issue with the low RIN.

For the samples today, I used a different method to avoid thawing the sample during weighing. First: 1ml of buffer can handle 30-50m of tissue. I know the weight of each tissue so I pre-allocated out enough buffer so that I have 50mg of tissue for each 1ml of buffer. I'm going to have to buy more buffer to be able to finish the kit, but that's not a huge deal. Second, each column will handle up to 100mg of tissue which means I will use 2ml of buffer to go on each column. This way, every time I finished grinding a placenta, the powder went straight into the pre-measured buffer without ever thawing, then I used 2ml of the buffer - often having plenty left over (especially for the overgrown crosses).

The next issue is that the powder is so cold that it will freeze the buffer. Once the buffer freezes, it forms a shell around the powder and since it is water-based it has a much higher specific heat than the powder. This means that the buffer shell will stay frozen as the powder inside thaws and degrades. This is a huge issue and the first 5 extractions (I've done 8 so far out of 40) may all be prone to this degradation issue.  To circumvent this one I am dumping the powder into the buffer instead of the buffer into the frozen powder tube. It helps a lot, but still isn't 100%. I'm going to run the bioanalyzer tomorrow morning to check them out and see how degraded everything is - hopefully not at all.

The miRNA kits are interesting. There are two separate columns: one for tRNA (the "t" here stands for "total" not for "transport"), and one for miRNA (micro RNA). You run everything through the tRNA column and then take the flow-through and put that onto the miRNA column. Then they tell you to put the tRNA in the fridge and finish purifying the miRNA before going back to finish the tRNA purification. This may make sense if you are more worried about the miRNA, but I really want the tRNA, the miRNA is really just an afterthought - we're not even sure what experiment to do with it, we just thought it might be nice to have in case we think of anything... suggestions are more than welcome.    But I wonder if another reason the RIN score was so low for the first sample was because I set the tRNA in the fridge for 30 minutes while I finished the miRNA section. It may be a better idea to put the miRNA one in the fridge and do the tRNA one first. I'm going to have to talk to J~ about this once I run the others through the bioanalyzer and see their RIN numbers.