RNA Extraction For qPCR Analyses

 Introduction:

    Previous experiments have been conducted using qPCR to evaluate the gene expression in Deinococcus radiodurans for six target genes including acylase. lactonase, Met H, Met K, pfs, Lux S, and two reference genes, secA and GAP3. After two previous runs resulting in gene expressions in the treatment groups statistically identical to the control groups, it was determined that the 50 mM hydrogen peroxide solution may not be enough to induce the level of oxidative stress desired to witness changes in the gene expressions. A new run with the same target and reference genes will be conducted, using 100 mM hydrogen peroxide solution at the same 30 minute duration as previous trials. The first step in this process is to conduct a successful RNA extraction from the three treatment groups and three control groups. I did this process from start to finish by myself for the first time in order for Chad Albert to continue the process with cDNA synthesis and subsequent qPCR. The goal was to retain enough RNA while ensuring the 260/280 and particularly the 260/230 values, which were an issue previously, were as close to 2.00 as possible for a successful reaction and accurate results. 

Methods:

     D. radiodurans was inoculated in TGY media by Chad Albert and allowed 24 hours for growth.  An OD 600 reading was taken from this culture with Nanodrop One, and each of the six tubes were individually adjusted for an OD 600 reading ranging from 0.95 to 1.01, diluted with TGY as needed. The cells were pelleted by centrifugation for five minutes at max speed, the supernatant extracted, and each pellet resuspended in DNase/RNase free water as a wash. The tubes were spun again, and supernatant disposed of. 200 microliters of 100 mM hydrogen peroxide solution was added to the three treatment groups, while the control groups were resuspended in 200 microliters of the same DNase/RNase free water used in the wash procedure. Each was homogenized by pipette, and flicked repeatedly to ensure no air bubbles were in the solutions. When they appeared identical, incubation was done at 30 degrees Celsius for thirty minutes. 

     RNA extraction began with centrifuging each tube and 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. The treatment and control samples were alternated and tubes centrifuged so 350 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 95% ethanol was added to the flow through and 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 consisting of 35 microliters of DNase 1 and 525 microliters of DNA Digestion buffer was made and mixed separately, then 80 microliters of this solution added to each column. The tubes incubated at ambient temperature 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:

    Figure 1 shows the values measured using the Nanodrop One, in which each sample has a volume of 50 microliters. The lowest value for ng/ul was treatment tube 1 with 292.2 and the highest was control tube three with 879.2. The 260/280 values were very similar across all samples, ranging from 2.08 to 2.11. The 260/230 values were slightly higher than achieved in the recent runs completed by Chad, but still an acceptable range from 2.10 to 2.26. 


Tube

ug/ul

260/280

260/230

T1

292.2    2.082.26

T2

472.42.102.20

T3

358.82.102.22

C1

609.72.112.10

C2

398.52.112.14

C3

879.22.082.20

Figure 1


Conclusions:

    These RNA samples were successfully acquired and are in the acceptable range for each type of value, ng/ul, 260/230, and 260/280, so that cDNA synthesis can proceed and a qPCR trial can be accomplished. The values indicating sample cleanliness were higher than previous attempts at completing this protocol, likely due to more inexperienced pipetting in comparison to Chad Albert, particularly the step that involves extracting 350 ul from the bead beating tubes, which was the most strenuous portion of the entire kit. The ng/ul values were also the highest seen yet during this experiment, potentially indicating that the 100 mM hydrogen peroxide solution elicited a strong response from the cells in comparison to the previous trials. Hopefully qPCR results will be seen this week from these samples.   

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