RNA Extraction from Deinococcus radiodurans Exposed to Oxidative Stress

 Introduction

    Previous tests conducted with qPCR done by myself and Chad Albert have verified that the acylase and lactonase primers that we possess have sufficient efficiency (within 90 to 110 percent) to begin tests evaluating how oxidative stress affects these gene expressions. Among acylase and lactonase there will be four other targets genes tested including Met H, Met K, pfs, Lux S, and two reference genes, secA and GAP3. Deinococcus radiodurans is significant because it produces both acylase and lactonase, when typically only one is expressed in any given bacteria. By completing this qPCR experiment, a comprehensive understanding of how D. radiodurans responds to oxidative stress will be possible. The preliminary steps to acquire this data will be to inoculate and allow for sufficient growth, then complete a successful RNA extraction from six samples in which half of these samples were exposed to oxidative stress. From these RNA samples, cDNA synthesis will be completed and qPCR can take place. 

Methods

    D. radiodurans was inoculated in TGY media by Chad Albert from a freeze back sample,  and given 24 hours to undergo growth. An OD 600 reading was taken from this culture with Nanodrop One, and it was diluted to reach and OD 600 reading of approximately 0.8. The dilution required initially 190 microliters of culture and 1.81 ml of TGY, but the OD 600 reading was still higher than desired so further diluted with 1.42 ml of the secondary solution of culture and 0.58 ml of TGY, which was successful. Six aliquots of 200 microliter samples, three treatment tubes and three control tubes were made. The three treatment tubes each received 100 microliters of hydrogen peroxide solution, mixed by flicking each tube, and all tubes incubated for one hour in the dark at 30 degrees Celsius. 

    A PBS wash was completed with 300 microliters of 1x PBS solution once tubes were centrifuged and cells pelleted, and supernatant disposed of. The pellets were resuspended in the PBS solution and tested for OD 600 readings to ensure enough cells in each sample to get enough RNA in the following steps. The treatment groups has an OD 600 between 0.3 and 0.4, and the control groups had 0.4 to 0.5. The tubes were pelleted by centrifugation and supernatant extracted an disposed. 

    RNA extraction began with resuspending the pellet in 800 microliters of RNA lysis buffer, and transferred into a ZR Bashing Bead lysis tube. The tubes were subjected to ten total minutes in the bead beater in one minute intervals of beating and two minutes of rest on ice to prevent detrimental hear exposure. The treatment and control samples were alternated and tubes centrifuged so 400 microliters of the supernatant could be carefully extracted and transferred into a Zymospin III CG column in a collection tube. The columns were centrifuged and the flow through was saved for following steps. Equal volume of 100% ethanol was added to the flow through an pipetted for thorough mixing. The total 800 microliters of each sample was transferred to  a Zymospin IICR Column in a collection tube and centrifuged for one minute. The flow through was discarded and 400 microliters of RNA wash buffer added to the column, centrifuged for one minute and flow through discarded. A DNase 1 reaction mix made of five microliters of DNase 1 and 75 microliters of DNA Digestion buffer per sample was made and mixed separately, then the total 80 microliters added to each column. The tubes incubated at 30 degrees Celsius for fifteen minutes. Then 400 microliters of RNA prep buffer was added to the column, centrifuged, and flow through discarded. 700 microliters of RNA wash buffer was added to each column, centrifuged, and flow through discarded. To ensure complete washing, another 400 microliters of RNA wash buffer added, centrifuged, and flow through again discarded. The column was transferred to a nuclease free tube, and 50 microliters of DNase/RNase free water directly added to column and centrifuged. The resulting sample was measured for RNA values with the Nanodrop One prior to cDNA synthesis. 

Results

     The initial OD 600 reading of the primary culture was 8.3. The RNA nucleic acid readings taken prior to cDNA synthesis are in Figure 1.

Tube	ug/ul	260/280	260/230
T1	23.0	1.95	0.77
T2	45.6	1.89	0.48
T3	38.0	1.91	0.60
C1	28.2	1.95	0.75
C2	113.4	1.79	0.45
C3	36.0	1.94	0.66

Figure 1

Conclusions

    The RNA nucleic acid readings did not supply sufficient confidence going forward, and it was determined the experiment should be redone in order to be sure that qPCR would be successful. The only tube that has a significant amount of RNA detected was C2, but every other tube was relatively low. This would have been potentially sufficient except for that the 260/230 values were so low that it made it incredibly likely that PCR reactions would be unsuccessful. These values indicated that they were too contaminated and the PCR reactions would be greatly inhibited. The 260/280 values were deemed sufficient. It is speculated that the potential problem, at least in terms of low RNA values, is that there was an unknown problem with the starting culture. The culture was discolored in comparison to typical pink color of D. radiodurans and was a more milky brown color. Chad Albert also believed the smell was strange, but when he did a gram stain of the sample he did not find any contaminants. It was also found to be odd that the initial OD 600 reading was 8.3 after only 24 hours of growth, when he typically sees OD values between 2 and 3. It was determined that a brand new culture should be inoculated, potentially from a plate with multiple colonies in order to give us the ability to choose which colony to inoculate, and start fresh with a culture we are more confident in. The 260/230 values may have been due to pipetting error, but it is not for certain at this point.