Week of 9/30/24-10/4/24

Intro

The entirety of this week was dedicated to RNA extraction, an RNA clean-up procedure on all of the samples, and running multiple RNA gels to verify results of said RNA extraction.

Methods

Note: control samples (D, E, F) were normalized the previous week, pelleted, and supernatant was removed before placing samples into -80°C freezer over the weekend to be ready for RNA extraction of control samples as well as test samples on Monday 9/30/24

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
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 directly to column matrix and centrifuge

RNA clean up procedure:

1. Add 2 volumes RNA binding buffer to each sample and mix
2. Add an equal volume of ethanol (95-100%) and mix
3. Transfer the mixture into a Zymo-spin IICR column in a collection tube in centrifuge for one minute. Discard the flow through
4. add 400μl RNA prep buffer into the column and centrifuge for one minute. Discard the flow through
5. add 700μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
6. add 400μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
7.  add 400μl RNA wash buffer into the column and centrifuge for two minutes. Discard the flow through
8. transfer the column into a nucleus free tube, and add 50μl DNase/ RNase free water directly to column matrix and centrifuge

RNA gel was made with a 1% TAE solution that was allowed to cool slightly before adding 2uL of midori green for visibility of RNA.

Results & Discussion

Results from the RNA extraction procedure are as follows:

(Samples are A-F, 1-6)
After the RNA clean-up procedure, results were as follows:

After running the RNA gel on 10/3/24, all of the samples were still intact except for sample F. A second gel was run just for sample F with the following results:
This confirms that the control sample F was degraded.

Conclusion
Based on these results, we have decided to continue with desiccation procedures to obtain more control & test samples that all show up cleanly on a gel before moving forward with sequencing. We hope that the next set will be our final set before being able to take that step forward.

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