Summer and Ref Gene recap

 Intro

    Over the course of the summer, it was decided that we should pivot away from the AMC project and to work on something new. Our focus shifted to selection and validation of reference genes for qRT-PCR analysis in Deinococcus sonorensis under the condition of desiccation. 

Method

    Most of out methods changed over the course of the summer. For example, at the beginning of the project we were rehydrating our desiccated cells with water, which we later changed to a rehydration buffer to avoid essentially popping the cells.

    In general the basis of the project is to desiccate cells, collect RNA from those cells (as well as a non desiccated control set), run RNA-seq on the collected RNA, identify differentially expressed genes and rank their stability through various programs, and finally validate the DEG's through qRT-PCR and qRT-PCR analysis.

Our original desiccation protocol is as follows:

1.  Grow cells for 48 hrs @ 30 °C on R2A media. 

2.  Resuspend colonies into 50 mL of R2B media

3.  Centrifuge cells at 1800 RCF for 10 minutes @ 4 °C  twice, then adjust OD600 to        0.8 ± 0.1

4.  Serial dilute suspension  and plate each dilution in triplicate and incubate @ 30 °C

5.  Plate 10 μl of the original (adjusted OD value) suspension into a 24 well plate in triplicate and incubate plate at 30 °C 

6.  Rehydrate wells from 24 well plate for 5 minutes in 300 ul of nuclease free water. Repeat step 3 for post-desiccation

Note: step 6 changed to be rehydrated w/ rehydration buffer (300 uL of buffer per 100 uL of spotted cells), then let sit on rocker for 15 minutes at 110 rpm 

D. sono desiccated cells after 24 hours of growth, diluted to 20x


D. sono non desiccated cells after 24 hours of growth, diluted to 20x

The RNA isolation procedure is as follows:

  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 10 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

Results & Discussion

    Even though the total amount of spots per well plate and the volume of each spot was increased, there were still issues with collecting a decent yield post RNA isolation. Often, there were problems with the cleanliness of the sample as well.


Values measured using Nanodrop


For the majority of the summer, we were working with sonorensis because of its relation to radiodurans, which is a known desiccator species. The hope was that by picking a sister species to radiodurans, we would have another desiccator on our hands. The issue with sonorensis, though, is that it produces a tough biofilm, making it very difficult to get the cells to break open, making RNA isolation a particularly tricky process. After multiple bouts of less than stellar results, it was decided that we would switch to Deionococcus deserti instead. As a species without biofilm and similarly close to radiodurans, hopefully deserti will be more effective for our uses in the long run. 

Conclusion

    As stated above, to avoid further isues related to species, we're pivoting to deserti for the rest of this project. Luckily with sonorensis we were able to fully set out a plan for the project as well as test and tweak procedures, so we aren't starting completely from square one. With deserti, we will need to test desiccation up to 7 days and see if it survives before moving forward full steam ahead as well as continue with the dilution series.

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