Week of 11/4/24-11/8/24

Intro

Between me being sick and Alex being sick later in the week, there was  limited amount of progress that was made this week. With our time, we did manage to get another set of RNA extraction done with some minor modifications to our normal procedure in order to hopefully increase our overall yield at the end so that we can move forward with sequencing.  

Methods

Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes.

RNA isolation procedure:

1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube
2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times without repetition (minor change)
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. Save the flow through
5. Add an equal volume of ethanol (95-100%) and mix well
6. transfer the mixture into a Zymo-spin IICR column in a collection tube in centrifuge for one minute. Discard the flow through
7. add 400μl RNA wash buffer to the column and centrifuge for one minute, then discard the flow through
8. prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube) and add 80μl directly into column matrix and incubate at room temperature (20-30°C) for 15 minutes, then proceed with purification
9. add 400μl RNA prep buffer into the column and centrifuge for one minute. Discard the flow through
10. add 700μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
11. add 400μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
12. add 400μl RNA wash buffer into the column and centrifuge for two minutes. Discard the flow through
13. transfer the column into a nucleus free tube, and add 50μl DNase/ RNase free water (heated to 60°C) directly to column matrix and centrifuge

Results & Discussion

Results for the RNA extraction procedure were as follows:

ng/ul

a260/280

260/230

A

68.6

2.21

1.36

B

22.02.20

1.87

C

65.9

2.13

2.02


With one of our samples not having enough yield and two of them needing to be put through the purification procedure, we would lose even more of our yield with no guarantee that we'd still have enough leftover in order to proceed with quality checks and still have enough for sequencing.

Conclusion

Based on our numbers we will be starting over again. After a discussion with Dr. Tuohy, we have been strngly encouraged to increase our amount of tests from 3 to 5 as we always have at least one sample that isn't good enough to be used, so with 5 as opposed to 3 we should hopefully be able to get around that while keeping our overall numbers in triplicate.

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