Today I tried out the new plan for extracting RNA. It took quite a long time and none of the 24 samples had detectable RNA. Details in post.
Set up and preparation
The longest part of this whole thing was labeling tubes.
- 24 RNase-free snap cap tubes (for the 15ul of slurry)
- 24 QIA shredder columns (cap and side of tube)
- 24 gDNA columns
- 24 RNeasy MinElute columns (had to do this right before use because they’re supposed to be cold… but it took forever, so they probably weren’t cold)
- 24 1.5ml snap cap tubes that contain the eluted RNA
I prepared solutions for 24 samples plus extra:
- 70% ethanol (7mL ethanol and 3mL DEPC-H2O)
- 80% ethanol (10mL ethanol and 2.5mL DEPC-H2O)
- BufferRLT Plus and B-ME (9mL Buffer RLT and 90ul B-ME)
Sampling out slurry
I selected 24 samples –> two from each of the 12 temp treatments/infection status groups
|Tube number||sample day||infection status||temp trtmnt|
This part also took a really long time. For one, finding the tubes in the -80 took some time because I did not place them in there in number order.
Additionally, it took a long time to let them thaw, vortex for a few seconds, and then sample out 15ul of the slurry.
Thawed hemolymph slurry:
Starting protocol finally (1.5hrs for 24 samples)
- Added 250ul of Buffer RLT + B-ME (did under hood in 209 because it smells awful)
- Vortexed all for a few seconds
- Transfered contents to QIA shredder columns (under hood as well because stinky)
- Centrifuge 2min full speed (takes a while putting in and taking out 24 tubes)
- Transfer flow-through to gDNA eliminator column with 2ml colletion tubes. Centrifuge 30s at full speed. Discard column. Save flow-through. (While this was happening, I was furiously unwrapping and labeling RNeasy MinElute columns… took a long time… samples sat in centrifuge for a few mins…)
- Add 350ul 70% ethanol (pipetted individually). Mix by pipetting.
- Transfer sample to RNeasy MinElute column. Close lids. Vacuum.
- Add 750ul of Buffer RW1 (used repeat pipet- amazing!). Close lid. Vacuum.
- Add 500ul of Buffer RPE (used repeat pipet). Close lid. Vacuum.
- Add 500ul 80% ethanol (used repeat pipet). I think I miscalculated my volumes in the preparation, because I ran out of it and had to run and make more. Close lid. Vacuum 5mins. (While vacuuming 5mins, I was labeling the 1.5ml snap cap tubes).
- Put RNeasy column in new 1.5ml snap cap. Add 14ul RNase free water to center of membrane. Cut off pink lids. Centrifuge for 1min at full speed.
Made working solution: 5.6mL Buffer for RNA HS + 28ul RNA HS dye
Made standards: 10ul of each + 190ul working solution
Ran 1ul of each sample (added 199ul working solution)
ALL TUBES READ “OUT OF RANGE, TOO LOW
Put the hemolymph pellets that I thawed and used 15ul of in -80 (Rack 7, col 2, row 2)
Put eluted “RNA” samples in -80 (Rack 7, col 3, row 1)