Hi there,
I'm looking to compare the expression of a particular gene in fish brains across three developmental stages. My aim (as first step) is to use the entire brain for RNA isolation steps. This is simple with larval and juvenile stages, however, the RNeasy Lipid Kit has a maximum tissue weight of 100mg. Thus, it is far less easy to extract RNA representative of the entire brain in adult fish (with brains that are coin-sized).
So far, I've tried grinding the adult brain in liquid nitrogen using a large mortar and pestle, mixing the cold 'powder', then weighing and using 100mg of it in the kit - hoping that this sample comprises bits of tissue from all parts of the brain.
I worry, of course, that I'm not truly getting a true representation of the entire brain when I do this. Does anyone have any suggestions on what to do differently to get the same result?
Thank you in advance!
What you are already doing sounds like a reasonable way to get even representation. To make it even better, you could prepare a liquid homogenate or lysate from the entire brain and then use a subsample of this liquid for your RNA extraction.
We routinely homogenise mouse brain tissue in PBS so that we have a liquid that evenly represents our brain region of interest that can be divided up for multiple experiments. We use glass dounce homogenisers and 500-1000uL of PBS for 15-50mg of tissue. I think you could probably 'fit' quite a bit more tissue into the same volume of PBS. When RNA quality is really really critical, we supplement the PBS with DTT (1uL/mL of 1M DTT) and RNaseOUT or Superasin (1uL/mL).
To maintain RNA quality, it is critical to keep your sample cold. We dissect on ice and store tissue samples at -80C. Tissue is thawed immediately before it is homogenised (it comes out of dry ice, into my hand just long enough to unstick the sample from the tube, and then directly into the homogeniser, one sample at a time) and solutions and equipment for homogenisation are kept ice-cold. Under these conditions we routinely extract RNA of RIN9+ from mouse brain.
Homogenate in PBS should be processed as a liquid sample (follow kit instructions for liquid sample when determining volume of lysis buffer etc.) Alternatively, after aliquoting the required amount of homogenate, you can centrifuge it at moderate speed (5000 * g) for 3-5 minutes at 4C and process the pellet as if it was tissue. I prefer not to do this for brain tissue because homogenising shears neuronal processes and these can end up in the supernatant, so you throw out some of the axonal and synaptic RNA.
Another option is to homogenise your tissue directly in lysis buffer, using the appropriate volume of buffer for the weight of tissue. Then, take an aliquot of lysate that suits your kit capacity, and process that. This is slightly better for your RNA quality, but will waste a lot of lysis reagent, which is why we don't do it (since the RNA quality with the PBS method is so high anyway.)
Hope this helps :)
Thank you so much for the thoughtful response Laura Leighton - that's exactly what I was looking for! I'll give those techniques a shot and see how they compare!
- Badges
- Science method