Thursday, November 19, 2009

Experimentation Complete

Filtering has finally been completed. In total we filtered ~91 liters of sample! Our next primary objective was to extract RNA from one of the filters and quickly convert it to cDNA to prevent degradation. We can then ship the cDNA back to LSU to sequence.

While challenging, the information we will learn from this type of analysis will be very valuable. Sequencing the 16s rRNA fragment directly allows us to determine which species of microbes have remained viable when trapped inside Taylor Glacier. The normal method of just sequencing the 16s rRNA gene (which is located on DNA, not RNA) only tells us which microbes were present in the glacier at one point in time. Any microbes which died in the glacier thousands of years ago would no longer be viable, but may have have left there DNA behind. In other words, with DNA alone, it is difficult to tell which sequences came from viable cells as opposed to those sequences which came from dead cells.

Our extraction turned out to be a success as we managed to extract ~25ng of RNA from one filter!!! From this RNA we reverse-transcribed the 16s rRNA molecules to cDNA which is now on it's way to LSU to be sequenced!

Additionally, we measured the amount of ATP in our samples with the luminometer (as described last month on the blog). This experiment was successful as well, indicating about 10,000 cells/mL. This reinforces some direct microscopic cell counts we did earlier which yield roughly the same amount.