Recently I took some time to travel back to Michigan to take care of some of our biological samples. As you may have heard we have updated how we handle DNA extractions from hyena blood. Originally we utilized an extraction method that I described in a prior post that required us to do extractions out in the open, and can be a bit stressful prior to shipping them home. Now we use tubes that either stabilize all the cells (DNA tubes) or shred all the blood cells in the tube and stabilize the nucleotides from white blood cells (RNA tube) for storage and transport back to the US. However, we still need to do the extraction process back in the US, but thankfully in a much more controlled environment. Once the samples reach the US with Kay they get stored in huge freezers, and logged into our frozen tissue database.
From these freezers, grad students and collaborators will pull their samples for a variety of projects, from fecal hormone analysis to RNA expression measures. In my case I am pulling our new samples to complete the DNA, as well as RNA extractions. I typically transport my samples down to the MSU genomics core, or to a lab space we are allowed to use thanks to a project collaborator Dr. Barbara Lundrigan.
|My lab space, that I am able to use thanks to Dr. Lundrigan|
|Photo thanks to the MSU Genomics core website:|
After thoroughly sterilizing the lab equipment and benches (it had been long enough since I had been in the lab that I bleached the bench, and wiped everything with 70% ethanol) we were able to start the extraction process. What followed, at least for the DNA extraction, was a series of what is best called washing the samples. After we defrost the sample from the field we dump the liquid into a big tube of “buffer", the first of which shreds red blood cells, and then spinning the tubes containing the samples in a very large centrifuge.
This leaves us a “pellet" of white blood cells, which is where the nucleotides are found (red blood cells don’t have a cell nucleus, which is where DNA is stored). This pellet is then washed with another buffer that shreds the remaining white blood cell walls, and the tube gets spun again to make a DNA pellet. Following this is a series of washes, and incubations, to ensure that any leftover proteins or contaminants are removed from the samples, leaving us with pure DNA. The RNA extraction is similar, except that the cells for these samples get shredded in tube used in the field, and back in the lab the nucleotides are spun out in the first round. However, there are many more cleaning steps involved for RNA extractions, including cooking the sample with enzymes that digest anything other than RNA. Following this we can check the quality of the samples on a variety of different pieces of equipment to determine how concentrated the samples are, and how pure they are. I prefer to use the NANO-drop in a lab down the hall from my lab space, and the Bioanalyzer in MSU’s genomics core.
|The genomics core Bioanalyzer work station.|
The "tower" on the right is the most expesive piece of equipment I've ever touched!
|First samples to ever be used for spotted hyena transcriptomics!!|
Once these samples have been prepared, people in our lab can do a variety of things. My mission while I was home was to measure gene expression of a particular gene called SERT, which helps modify serotonin levels in the body, and to build the spotted hyena blood transcriptome via RNAseq. A transcriptome is basically a measure of what genes are actively being transcribed within a tissue, and RNAseq is a technique that allows you to not only detect which genes are turned on in that tissue, but also how much. With enough RNAseq samples you can even tell how all genes vary in expression between individuals, but it is much more expensive than the techniques used for single gene studies, which is why I am still using simpler methods for measuring the SERT expression. Thanks to the staff in the MSU genomics core I was able to get everything submitted, before hoping back on a plane for Kenya, and now I’m back with all of my cubs, whom of course don't look the same anymore, or have graduated from den dependence while I was away……….
Needless to say I am very grateful to the RAs in the field who have been very patient in helping me relearn all the spots of the cubs that grew up while I was away, and the staff in the MSU genomics core whom were willing to help with my frantic submission of samples.
Until next time