segunda-feira, 31 de março de 2014

Working hard on cDNA synthesis

Today, we, Maithê and Paula, will tell you a little about what we do with the collected samples of Loricariidae when they arrive in the lab. As previously written, after fishes has been collected and livers extracted, it's necessary to extract RNA and DNA. What do we do next with this genetic material?

Well, while the DNA is sent to the Paulo Buckup's team, our collaborators at the National Museum of Natural History, the RNA is used for synthesis of complementary DNA (cDNA). But what is cDNA, how is it done and serve for??
The cDNA is a strand of DNA complementary to a RNA molecule and made by the aid of an enzyme called reverse transcriptase. To make this happen, we need: 1 - buffer solution, to maintain the ideal pH; 2 - reverse transcriptase, which is the enzyme that will do the reverse transcription of RNA to cDNA ; 3 - dNTPs, the nucleotides used as substrate by the reverse transcriptase to synthesize cDNA; 4 - the RNA sample, that serves as a template for the reverse transcriptate; 5 - and primer to help the reaction start. We used anchored oligo dT primers.Those are a sequence of 18 to 25 Ts that bind to the poly A tail of messenger RNA (mRNA).
These reagents are present in the AppliedBiosystems' kit ''High Capacity cDNA Reverse Transcription Kit" and they are put together in ideal proportion, according to the manufacturer's protocol for cDNA synthesis, that occurs at 37 °C during 120 minutes.

So this is another step needed to achieve our goal. We hope you have enjoyed. Keep up with our blog !! Soon, we are going to write more about our adventures in the Lab.

Um comentário: