Results for PFS Gene Expression When Introduced to Oxidative Stress

     This week in lab, we began with starting over with our experiment. Last week, our RNA results came out very low and dirty, most likely due to improper handling of samples. For this experiment, we are looking to see if the gene PFS in Deinococcus radiodurans is expressing when put under oxidative stress with hydrogen peroxide. 

    We started growing our samples on Monday for a total of 24 hours of growth. Tuesday, we started the dilution process. We ensured all 6 samples were individually measured for 1 OD600. After diluting our samples, we the introduced 3 of our 200uL of D. rad. in TGY samples to 200 uL of 200mM of H2O2 for 30 minutes. Realistically the H2O2 was at 100mM due to it being diluted once it was added to the samples. For our controls, we just added 200uL of TGY for consistency in volume. Once incubation was completed, the cells for all the samples were washed thoroughly with TGY and pelleted for RNA isolation. 

    RNA isolation and cDNA synthesis took place on Wednesday. Only 1 member was to do each protocol to ensure consistency within pipetting and to limit contamination. After RNA isolation, our numbers were as such:

T1, T2, T3
C1, C2

C3

    Based on these numbers we were able to determine that these samples, although low in concentration, are significantly cleaner than our past RNA isolation attempt. The low numbers could very well be due to the fact that there are 6 samples that RNA isolation is being performed on. When starting the cDNA synthesis, we had to normalize to our lowest concentrated sample. That sample being T1. We put the max amount of RNA for T1 (14uL) into the cDNA reaction which ended up being 92.4ng total. Every sample was then done so the input was around 92ng.

T1 14uL - 92.4ng

T2 5.7uL - 92.9ng

T3 2.8uL - 91.28ng

C1 2.6uL - 91.78ng

C2 3.8uL - 91.96ng

C3 3.9uL - 93.21ng

    We followed the same protocol as usual for cDNA synthesis. After cDNA synthesis, we were then ready to plate. For our plate, it was laid out as such:

For our NT Controls, there was no need to do triplicate, but we did it anyways

    The first time we plated our qPCR, there was an issue with the seal and all the sample leaked when it was centrifuged. We tried again the next day. We did a 1/10 dilution of our cDNA so 5uL cDNA sample to 45uL nuclease free H2O for all our samples. For actual plating, we did 6uL of Master Mix of the respective gene and 4uL of the respective cDNA per well. We centrifuged the plate for 5 minutes then put it in qPCR to run. These were the results: 

Melt Curve

Quantitative Results 

    Based on these results, we were able to determine that PFS did not express any more than usual when put under oxidative stress. We are now shifting to target 2 different genes. Those genes are LuxS and Kat A.