RNA isolation and Alcohol Precipitate

 

Last week we decide against continuing with pfs and instead using both LuxS and KatA. While we didn't get to qPCR this week, I did get to perform RNA isolation by myself and and do a new procedure: alcohol precipitate. 

We started the week on Monday with a graham stain. All our cells looked good, and we had a ton of D.rad to work with. 




Running the RNA isolation on my own was difficult. There was alot of back and forth, and i was constantly leaving and re-entering the hood. Deja was able to arrive in time to help me for the last few steps, but for the first hour and a half I was doing what we usually have two people do.

Since we do the RNA isolation in the biological hood, I was very worried about contamination or even just forgetting a step trying to do everything. 

I did have a hiccup during one of the steps. I had been given instructions to try to do everything possible in the -20 ice box, instead of keeping it in normal ice when possible but doing normal steps in the hood in a tube holder. The idea was to keep the sample cold and keep the RNA from degrading as much as possible. After the 30 minute step where the sample was in the bead beating tube, I put the sample in the box like I was instructed in order to get it back to a cold temp. After about a minute, I moved on to extracting the samples from the tubes but discovered that the sample had already frozen and mixed with the beads. It was a simple fix; letting the tubes thaw again before trying to extract the sample.

After talking with Dr Tuohy, and than Chad, it was decided that while keeping the samples cold was a major part of the procedures; freezing and thawing the samples continuously in order to work with them posed a much larger risk to the RNA than working with it at a higher temperature. 

After that discussion I continued on with steps like we normally did: in the ice when possible but otherwise in the hood. The rest of the procedure went as smooth as possible with me moving in and out of the hood.  While I was worried about contamination and ruining the samples, we actually got the best results we've had so far. 

Most of the results from last week were around 20 or 30 ng/μl, this time most were around 110 ng/μl. 

       ng/μl      A260/280   A260/230
T1  147.4      2.09            2.09   
T2  177.6      2.06            1.63
T3  107.8      2.08            2.08
C1  111.5      2.11            0.40
C2  455.8     1.79             0.58
C3  570.9     2.06             1.79

While C2 and C3 had really high ng/μl concentrations, both the A260/280 values and Chad assured they were actually RNA. What was concerning was the A260/230 values for C1 and C2. The concentration and A260/280 were good, we just needed to get the A260/230 higher. We decided to do an alcohol precipitate. While it would most likely lower the concentration it would increase the A260/230 values enough to move on to cDNA synthesis. 


I started the alcohol precipitate Friday with Johnathan's help (it was a new and pretty precise procedure I didn't feel comfortable doing on my own, and with no-one in my group there to help me, he offered to.) 

We started by looking at a few different procedures. They seem to vary species to species, and there are different steps depending on what your project in general is. We also had to accommodate for what we actually had the ability to do in the lab (most of the procedures said to centrifuge at 4°C, but that wasn.t possible in our lab). In the end we decided on this procedure:


We started by taking 25 μl of our 50 μl post RNA isolation sample

We then added a tenth volume Sodium Acetate (2.5 μl) and two and a half times volume of 70% ethanol (62.5 μl)

We also decided to add 1 μl of glycoblue in order to see the RNA pellet in later steps. 

After vortexing, we placed the tubes into the -80°C freezer for exactly one hour.

After removing the sample from the freezer, we centrifuged at room temp and top speed for 15 minutes.

Next we washed the pellet in .5ml 100% ethanol, spun for 10 minutes at top speed, and removed any supernatant from around the RNA pellet

After repeated that step again, we let the pellet air dry for roughly 5 minutes in the hood. 

When all the ethanol remaining has evaporated, but the pellet hadn't lost moisture we resuspended it in 25 μl of nucleus free water. 


We nanodropped after the alcohol precipitate and found that our procedure had worked. While the concentration had gone down that was expected, and our A260/230 values improved. 

       ng/μl      A260/280   A260/230
C1  123.5      1.90            1.61   
C2  145.4      1.88            1.61


In the end, the alcohol precipitate was successful, and allows us to now move onto cDNA synthesis.

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