Evan’s Capstone – eNotebook 5

Wednesday 2/10/2016 and Thursday 2/11/2016

            Double update today since I didn’t have time to write up my report yesterday night and figured it would be simple to fill it in here! Wednesday was relatively simple, just finished up the final 7 RNA Isolations while today I worked on learning how to conduct proper DNAsing of the samples to prepare for reverse transcription and then qPCR.

Wednesday’s RNA Isolations:

3BRC and 9BRC (Rock Creek) in addition to 7BCO and 17BCO (Coulter Creek) were pulled at 12:20 PM to be used as the first set. The second set consisted of 1BRC and 12BRC with 19BCO and were pulled at 12:55PM. Samples were returned to the -80 freezer at 4:05 PM.

Only one slight deviation from the standard procedure that day and it was that Set 1 rested on ice after step 15 for 10 minutes while waiting for Set 2 in the centrifuge.

Nanodrop Results:

Example: X-B-SampleSite – 260/280: ###, 260/230: ###, Concentration ### ng/uL
1BRC – 1.82, 1.01, 436.4
3BRC – 1.95, 2.20, 1393.7
9BRC – 1.97, 2.14, 1279.0
12BRC – 1.91, 2.20, 872.8
7BCO – 1.92, 2.05, 611.0
17BCO – 1.92, 2.08, 766.1
19BCO – 1.91, 2.28, 925.0

Thursday’s DNAsing:

Relatively simple to learn, it went smoothly as far as I can tell other than some of the samples having lower post-DNAse nanodrops than before. The procedure was the same as what is listed in the Common Lab Protocols except on the step where on step where you add the deactivation reagent and incubate it, we let it incubate for 5 minutes as per the manufacturer procedure.

The first set of isolated samples was pulled at 12:05 PM and consisted of 3BMA and 5BMA in addition to 13BHA and 14BHA. As a note, 14BHA was the sample that only had 25 uL total of isolated sample because of a small liver sample size. This led me to adapt a procedure and dilute it down to 40 ng/uL as compared to the normal 200 ng/uL for every other sample concentration since its initial concentration was only 180 (with only about 20 uL when it calls for about 44). The second set of samples consisted of 6BRC and 7BRC (Rock Creek) with 11BSW and 13BSW (Swamp Creek) and were pulled at 2:05 PM.

Nanodrop Results Post-DNAse (same format):
3BMA – 1.97, 1.47, 109.6
5BMA – 1.96, 1.32, 125.8
13BHA – 2.01, 1.75, 111.1
14BHA – 1.89, 1.04, 28.3
6BRC – 1.97, 1.68, 107.8
7BRC – 1.99, 1.60, 111.8
11BSW – 1.91, 1.43, 113.5
13BSW – 1.98, 1.55, 130.3

Samples were returned to the -80 freezer in a new storage container (to signify they were DNAsed) at 3:55 PM for Set 1 and 5:30 PM for Set 2. It appears that a lot of concentration (and 260/230 values) were lost in the process of DNAsing, given that the 260/230 numbers normally decrease with concentration this was expected and given how little actual RNA is in 14BHA I am not surprised it only has a value of 1.04. The other samples were okay, was really wanting more in the 1.60 range but the 1.4+’s are okay too. Most of the samples lost a lot of concentration to DNAsing which was also expected since liver tissue samples are normally pretty high in DNA and RNA and then by removing the DNA it would make sense for the concentration to dip down more than other types of samples. Overall a good learning experience and the samples are likely still viable. Next day in lab will likely be better and I should be able to get all the other samples done if I work efficiently!

 

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