quinta-feira, 24 de outubro de 2013

Nucleic acids extraction journey

The field collection is over but fun continues in the lab! Here, in the lab, is here we spend most of our time. After sampling fish tissue, the next step is to extract the total RNA content of each individual sample. In 6 days, our brave and hard working undergrad students, Paula and Maithê, have extracted RNA and the DNA from 88 fish livers.

Maithê (left) taking tubes out of the centrifuge with Paula observing at the back. Paula (right) dropping the supernadant out of the tubes to resuspend the RNA pellet.

The RNA will be used in our project. It is from these molecules that we will be able to sequence our favorite genes, CYP1A and AHR. The DNA will be sent to our collaborators at the National Museum of Rio de Janeiro for phylogenetics studies. At an appropriate moment, these two pieces of data will come together and eventually clarify some of the ecological roles of AHR/CYP1A in the evolution of loricariids.

RNA extraction is tricky! RNAses, enzymes that degrades RNA, are omnipresent! They are just every and anywhere; on your fingers, on your hair, on your saliva, on the dust (oww there are tons of that on the dust!), air etc! If you don’t take the necessary precautions, your sample will get contaminated, your RNA degraded and your time & patience lost. The thing is that you just get to know whether your RNA preparation is good or not at the very end of the process, when you run your sample in an agarose gel and actually see the RNA. Well truly you don’t see the RNA but instead the light emitted by a molecule, ethidium bromade, attached to it. The expected (or the good) result is to see two bands on the gel upon illuminating it with U.V. light. These two bands correspond to two of the ribosomal RNA molecules, the 18S and the 28S. Now, guess what did we get? No, I am not giving the answer! Check the photo of some of our extractions bellow and get to your own conclusions!

Great job girls! Very well done. Now, let’s keep the hard working and start cDNA preps, PCRs, cloning and sequencing. Well, that if the supplies arrives but this is story for another post.

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