Isolating RNA from hemocytes

Oct. 30, 2020

Samples:

Animal_ID Condition Sex Stage Location
3 ambient F mature/spent Rack 6, col 2, row 2
6 ambient F ripe Rack 6, col 2, row 2
7 ambient F late active Rack 6, col 2, row 2
015 low pH F early active Rack 6, col 2, row 2
025 low pH F late active Rack 6, col 2, row 2
053 ambient F early active Rack 6, col 2, row 2
057 ambient F ripe Rack 6, col 2, row 2
060 ambient F early active Rack 6, col 2, row 2

RNeasy Protocol Modificaitons:

  • Sample notes:
    • These samples were supposed to be pelleted hemocytes from hemolymph samples, however pellets were not really visible in most samples
      • this is not totally surprising because they are usually transparent/translucent cells anyways.
      • Some samples had some other tissue type in them
      • 015 has a green pellet
      • 025 had a yellow pellet
  • generally following what I did yesterday https://shellytrigg.github.io/380th-post/
  • add BME to lysis buffer
    • Made 7mL RLT + 70uL BME (-20C 2nd shelf drawer 7)
  • Added 350uL RLT + BME to pellets, vortexed and pipetted up and down to mix
  • Pipetted lysate into Qiashredder colums and spun at max speed for 2 min.
  • Transfered lysate to gDNA eliminator column and spun for 30 seconds at 11000 rpm
  • Saved 180uL of homogenized lysate and froze at -80C in hemolymph box (Rack 6, col. 2, row 2)
  • Followed protocol for purification of total RNA
    • Added 270uL of 100% ethanol to ~180uL homogenized lysate and transfered to minElute column
    • spun at 11000 rpm for 15 sec.
    • Added 500uL buffer RPE and spun at 11000rpm for 15 sec. Repeated this step for a total of 3 times
    • Added 500 uL buffer RPE and spun at 11000 rpm for 2 minutes
    • dried the RNeasy minElute column by spinning at max speed for 5 minutes with the lids open and using a new collection tube
    • eluted RNA in 15uL RNase free H2O by spinning at 11000 rpm for 1 minute
    • I saved the columns

Quantification

  • I measured the concentrations of the RNA on the nanodrop because the Qubit was being used.
  • *NOTE: Not all Sample IDs in image are correct and samples are listed as follows:
    • H2O
    • 053_2
    • 060_2
    • 015_2
    • 7_2
    • 025_2
    • 057_2
    • 6_2
    • 3_2
  • eluted off the RNeasy minElute columns again with 15uL of warmed RNase free H2O (~37C) into tubes labeled with just the sample numbers in Sharpie.
  • All RNA samples were stored in the -80C box Rack 6, Col. 2, Row 2
  • discussed these results in Science hour and decided to check qubit concentrations because nanodrop can be inaccurate when RNA is at low concentration
  • Also came up with a plan to check samples for RNA by doing a qPCR on the RNA

Nov. 3, 2020

Quantification of Oct. 30 RNA

  • thawed RNA on ice
  • checked 1uL of each sample
  • all samples are too low for quantification
    • Standard 2 = 10.4ng/uL (should be 10ng/uL)

RNeasy Protocol Modificaitons:

I decided to go back to the homogenized lysates that I save on Oct. 30 to see if I could get any RNA from them.

  • I repeated the homogenization and gDNA elimination steps to rule out too much tissue or clogged columns
    • I thawed homogenized lysates at 37C for 4 minutes
    • gave them a quick spin
    • added 350uL RLT buffer (no BME)
    • Pipetted up and down to mix and transfered to Qiashredder columns, spun at max speed for 2 minutes
    • Transfered lysate to gDNA eliminator columns and spun for 30 seconds at max speed
    • Followed same protocol as Oct. 29:
      • Added 530uL 70% EtOH (since I had 350uL of lysis buffer + 180uL of homogenate), pipetted up and down to mix and transferred to minElute columns. Spun at 9000 x g for 15 seconds, discarded flow-through
      • Added remaining homogenized lysate to column, spun at 9000 x g for 15 seconds and discarded the flow-through
      • Added 700uL of buffer RW1 to minElute column and spun for 15 sec at 9000 x g, discarded flow-through
      • Added 500uL buffer RPE to minElute column and spun for 15 sec at 9000 x g, discarded flow-through
      • added 500uL 80% EtOH to column, spun for 2 minutes at 9000 x g, and discarded flow-through.
      • Dried column by placing it in new collection tube, and spinning for 5 minutes on max speed with the column lids open
      • Eluted with 20uL RNase free H2O prewarmed to 65C for 10 min on the bench. Then spun at max speed for 2 minutes.

Quantification

  • checked 1uL of each sample
  • all samples are too low for quantification
    • Standard 2 = 9.92 ng/uL (should be 10ng/uL)

Conclusions

  • It seems these samples may not have had very many cells at all since there is no RNA, because I was able to successfully isolate RNA from an old hemocyte sample from Nov 2018 but not these samples.
  • My hunch is that these hemolymph samples are very variable and I’m don’t feel confident they contain sufficient hemocytes to do this experiment.
  • I still have the “lymph” part of these samples, but if it’s just seawater then they’re a little useless.
    • I wonder if there is a way to tell, or if these could be used for the SRM assay Emma developed?
  • I also still have siphon, ctenidia, and gonad for these animals so it’s still possible to do develop qPCR on gonad tissue, but the whole idea was for this non-lethal sampling.
  • Will consult with Steven and the group on how to proceed