RNA Isolation and cDNA Synthesis on Deinococcus Radiodurans

             This week in lab, we did RNA isolation on deinococcus radiodurans. The reason for doing this is so we could synthesize cDNA. cDNA is a template we are using to study gene expression in D. rad when it’s exposed to oxidative stress. For now, we are doing qPCR for our primers on the gene Sec A to see if it’s a good reference for our experiment. 

            On Monday, D. rad was inoculated into 250mL of TGY media as well as three TGY agar plates. The broth is being used for RNA isolation and the plates for freeze back. On Tuesday, a gram stain was performed on the inoculated TGY media and determined that there was no contamination and RNA isolation was ready to be performed. 3mL of our sample was spun down into a 1mL Eppendorf tube. The process was performed as such:

 

D. rad inoculated in TGY media

-Add 1 mL of inoculated TGY to Eppendorf tube, spin down for 1 min.

-Remove supernatant without disturbing pellet.

-Repeat this process 3x total

 

            After the last time centrifuging sample, we then resuspended the pellet and nano dropped for the OD600 value. The OD value of the sample was 1.70 OD600. We then diluted the sample down so we ended up with an OD value of 0.63 OD600. This is within our target range of 0.60-1.00 OD600. Our next step was to the RNA isolation protocol:

 

 

 

RNA Protocol

DNase Protocol

 

 

 

 

There were some modifications to the original protocol due to it being the most efficient way of isolating our RNA. After completing our RNA isolation, we nano dropped our sample. It ended up being:

285.9 ng/μL  2.10 A260/A280  1.74 A260/A230

 

The next step is to convert the D. rad RNA into cDNA. This also includes a process in which any genomic DNA that was carried over into our RNA tube is removed. The DNase Master Mix is as follows:

 

     DNase Master Mix

iScript DNase	0.5μL
iScript DNase Buffer	1.5μL
Total Vol.	2μL

 

            The recipe for our first reaction for removing genomic DNA from our sample is:

2 μL DNase Master Mix

14μL RNA Sample

            The sample was then put through PCR for DNA Digestion as well as DNase Inactivation. After removing the genomic DNA, we were able to continue to cDNA synthesis. The recipe for cDNA Synthesis is:

 

iScript Reverse Transcription Supermix	4μL
DNase-treated RNA Template	16μL
Total Volume	20μL

 

            Finally for the last step, we put the sample through a final round of PCR to prime, reverse transcript, and finally RT Inactivation. The results came out great and the cDNA is ready to go through qPCR for primer efficiency. This will be done next week.