Pre-desiccation to RNA isolation Procedure

Introduction

This blog is both for the full week Nov 18-22 as well as the three days before thanksgiving break. We were able to do a full pre-desiccation to RNA isolation procedure as well as help with a number of various procedures in the lab. This Blog is covering the pre-desiccation to RNA isolation procedure.

Procedure

We started with our pre-desiccation on monday. From our flasks A B C D and E made last week, all except C were clear of contamination and able to go through desiccation. C was a yellow color with clear physical differences compared to the other flasks, as they all had the usual clumping and normal off-white color. We continued to pre-desiccation with the four flasks. 

1. Normalize 3ml of culture to an OD between 0.95-1.00

2. Spin down 1ml of media, remove supernatant and resuspend pellet in nuclease free water

3. Resuspend pellet, then centrifuge and remove supernatant

4. Add the second ml of culture, repeat washing steps

5. Add the third ml of culture, repeat washing steps

6. Plate 100ul dots into 1 inch kapton squares in a 6 well plate in triplicate

The two plates were left in the hood overnight to dry, but were still mostly hydrated 24hrs later. After another 24 hrs of drying they were moved to The Egg for desiccation. after 48 hrs they were ready for RNA isolation. As we usually do 6 kaptons on one plate and 3 in the other, we dont mark the bottoms. This was an oversight as they were not marked when both had 6 kaptons in them. As they were placed in the Egg without lids, we were unable to tell the A+B plate from the D+E plate. We put the lids back on the plate we thought they would go with but we are unsure.

We started with rehydrating with 650ul of the rehydration buffer, then shaking for 15 minutes. After that a cell scrapper was used and the cells were gathered. After centrifuging they again had a weird white layer on top from the rehydration solution. We then proceeded with the RNA isolation.

We continued with the two rounds of bead beating as well as warming up the elution water to 60 degrees using the thermocycler before the final step

RNA:

1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube

2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 2 cycles of 1 minute on and 2 minutes on ice

3. Centrifuge the tube for one minute to pellet debris

4. Transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Retain the flow through

5. Add an equal volume of ethanol (95-100%) and mix thoroughly

6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Discard the flow through

7. Add 400μl RNA wash buffer to the column, centrifuge for one minute, and discard the flow through

8. Prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube), add 80μl directly to the column matrix, and incubate at room temperature (20-30°C) for 15 minutes before continuing purification

9. Add 400μl RNA prep buffer to the column and centrifuge for one minute. Discard the flow through

10. Add 700μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

11. Add 400μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

12. Add 400μl RNA wash buffer to the column and centrifuge for two minutes. Discard the flow through

13. Transfer the column to a nuclease-free tube, add 50μl DNase/RNase-free water directly to the column matrix, and centrifuge

	ng/ul	260/280	260/230
A	33.4	2.13	1.98
B	25.8	1.99	1.41
D	27.0	2.01	2.06
E	29.4	2.05	1.10

After this, the project has been momentarily paused until a Chad and Dr. Tuohy are able to have a discussion. However the week after thanksgiving break we will be doing A-E controls just in case.

Discussion:

After other groups success with less bead beating time, and very high yields of RNA we also lowered our bead beating time yet saw no difference except all of our results were of low yield as opposed to one out of three. Heating the Elution solution was also supposed to help with the binding efficiency and make it so that less RNA is lost. 

At this point the kit is new from the one previously used, and the procedure is the same used as other groups with great success. The only things different are the heated water which was added these last two isolations, and the desiccation/rehydration solution. Yet our yield results seem to get worse with every isolation. We will continue to look into the procedure, but something that has been suggested is doing a run when rehydration with only water as the rehydration buffer has changed from the summer when we first tested it. 

Beyond the procedure itself, during this RNA isolation we had an issue with the centrifuge. During step 6, the samples were put in the centrifuge and adjusted like normal so they would be properly balanced, then started for a 1min spin. It spun for about 30 seconds then made a robot shutdown noise, sounded like it was shaking on the inside a bunch, fully started shaking then smelled like smoke. It flashed an error message later found to mean the rotor was over heating and the machine shut itself down. We were unable to open it, even physically and restarting the machine a number of times wasn't enough to reset the heat lock. It was a few minutes later when we were able to get our samples out and even though we immediately continued the procedure, they still sat in the heat for a few minutes. This most likely impacted the RNA and could be a reason the yield was low, or the reason for the high level of organic contaminates in two of the samples although that would be less likely as the other two were fine on contaminates.