RNA Isolation

 This week was a short week for me as I was sick, with RNA isolation and normal project supplies upkeep being the only things done as well as general assistance of other groups.

Monday we performed RNA isolation. The samples were grown and prepped last week, and had undergone 2 days of dehydration and 3 days of desiccation. 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. We also heated the nuclease free water to dilute in to 60 degrees Celsius to help with the binding efficiency and make it so that less RNA is lost. 

Heating elution can significantly improve RNA yield through several key mechanisms. Higher temperatures help break down complex RNA secondary structures, increasing the molecule's solubility and making it easier to extract from binding surfaces. By partially denaturing RNA, heat reduces hydrogen bonding and disrupts potential interactions that can trap RNA molecules on membranes or extraction surfaces. The increased molecular kinetic energy facilitated by heating helps overcome binding forces, allowing more efficient detachment of RNA from columns or magnetic beads. Additionally, heat slightly reduces elution buffer viscosity, which improves liquid movement and recovery efficiency.

Carefully controlled heating can actually protect RNA by disrupting potential RNase interactions and reducing the time RNA is exposed to room temperature conditions where degradation can occur. Typically, researchers use elution temperatures ranging from 55-65°C, carefully balancing the benefits of heat with the need to prevent RNA degradation. The goal is to optimize RNA extraction by using temperature as a strategic tool to enhance solubility, accessibility, and overall yield while maintaining the molecular integrity of the RNA molecules. The process works by essentially "loosening" the RNA from its binding sites, making it more likely to be released into the elution solution. This approach is particularly useful in RNA extraction protocols like ours where maximum yield and purity are critical.


Procedure

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

a260/280

260/230

A

68.6

2.21

1.36

B

22.02.20

1.87

C

65.9

2.13

2.02



At this point one of our samples seems to be consistently lower than the others, with our minimum needed to be 65ul. This time I even swapped the order of the samples during the procedure as i usually go A B C to make sure that wasn't the cause. A discussion with Dr. Tuohy is making us consider switching to 5 biologicals that way even if one is worse, we still have 3 to run sequencing with. 

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