meth_3.2.2 Qiagen RNeasy PowerSoil Total RNA Kit modified for marine sediments Marine Microbes Coastal Project

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Extract RNA from frozen sediment samples (2 g) with Qiagen RNeasy® PowerSoil® Total RNA Kit (https://www.qiagen.com/au/products/discovery-and-translational-research/dna-rna-purification/rna-purification/microbial-rna/rneasy-powersoil-total-rna-kit/?clear=true#orderinginformation) following the modified extraction protocol below.

  1. Prep a 15 mL Bead Tube with 2.5 mL of Bead Solution, 0.25 mL of Solution SR1 and 0.8 mL of Solution SR2.
  2. Add 2 g of sediment

*Weigh 6 samples, then continue.

Move to FUME HOOD

  1. Add 3.5 mL of phenol:chloroform:isoamyl alcohol to the bead tube, cap tightly and vortex to mix until the biphasic layer disappears.

*All waste needs to be kept in fume cupboard and double bagged in a hazardous waste bag.

*Double glove and change gloves any time working outside of the fume cupboard. All pipettes etc. to be ethanol wiped at the end of use.

*Once phenol:chloroform has been added for 6 samples, start Step 4: vortexing as this takes 15 min.

  1. Vortex in vortex adaptor at maximum speed for 15 minutes.

*While these are vortexing, weigh other 6 samples.

*Start preparing 2 sets of collection tubes with 1) 1.5 mL of Solution SR3, 2) 5 mL of Solution SR4.

  1. Remove the Bead Tube from the Vortex Adapter and centrifuge at 2500 x g for 10 minutes at room temperature.
  2. Remove the Bead Tube from the centrifuge and carefully transfer the upper aqueous phase (avoiding the interphase and lower phenol layer) to the 15 mL Collection Tube with 1.5 mL SR3, vortex to mix.

*Avoid tilting the tube.

*MAKE SURE NO PHENOL:CHLOROFORM IS TRANSFERRED before adding to SR3. If

interphase is punctured, centrifuge again for 10 min.

  1. Incubate at 4 °C for 10 minutes. Wait until last batch has incubated to catch up.
  2. Centrifuge at 2500 x g for 10 minutes at room temperature. Transfer the supernatant, without disturbing the pellet (if there is one), to the 15 mL Collection Tube with 5 mL SR4.
  3. Invert or vortex to mix, and incubate at room temperature for 30 minutes (choose a cool place in the lab, as heat is not good for RNA – desk cupboard).

*Time to start thinking about LUNCH 🙂

  1. Centrifuge at 2500 x g for 30 minutes at room temperature.

*In the last 10 minutes of centrifuging, RNAseAway and ethanol treat biological safety cabinet. Get waste beaker and lay out kimwipes to UV sterilise. Any racks, pipettes, open pipette tips that will be used can also be sterilized in the cupboard.

FROM NOW ON IN STERILE BSC

  1. Decant the supernatant and invert the 15 mL Collection Tube on a paper towel for 5 minutes. Watch that the pellet does not drop onto kimwipe. This may happen if the sample was left to sit for a while after centrifugation.
  2. Shake Solution SR5 to mix. Add 1 mL of Solution SR5 to the 15 mL Collection Tube and resuspend the pellet completely by repeatedly pipetting or vortexing to disperse the pellet.

*Depending on the soil type, the pellet may be difficult to resuspend. Resuspension may be aided by placing the tubes in a heat block or water bath at 45 °C for 10 minutes, followed by vortexing. Repeat until the pellet is resuspended.

  1. With new RNAse free gloves, prepare one RNA Capture Column for each RNA Isolation Sample:
  2. Remove the cap of a new 15 mL Collection Tube and place the RNA Capture Column inside the 15 mL Collection Tube. The column will hang in the 15 mL Collection Tube.
  3. Add 2 mL of Solution SR5 to the RNA Capture Column and allow it to gravity flow through the column and collect in the 15 mL Collection Tube. Allow Solution SR5 to completely flow through the column.

*DO NOT ALLOW THE COLUMN TO DRY OUT PRIOR TO LOADING THE RNA ISOLATION SAMPLE.

  1. Add the RNA Isolation Sample from Step 12 onto the RNA Capture Column and allow it to gravity flow through the column. Collect the flow through in the 15 mL Collection Tube.

*When there is time, with new RNAse free gloves, start carefully capping and labelling 2.2 mL collection tubes from the kit. Add 1 mL of SR4.

  1. Wash the column with 1 mL of Solution SR5. Allow it to gravity flow and collect the flow through in the 15 mL Collection Tube.
  2. Transfer the RNA Capture Column to a new 15 mL Collection Tube. Shake Solution SR6 to mix and then add 1 mL of Solution SR6 to the RNA Capture Column to elute the bound RNA into the 15 mL Collection Tube. Allow Solution SR6 to gravity flow into the 15 mL Collection Tube. Do NOT throw out Capture Column.

*PREPARE 15mL DNA COLLECTION TUBES. After the SR6 has completely flowed through the column, transfer the Capture Column to the DNA Collection Tubes.

  1. Transfer the eluted RNA to the 2.2 mL Collection Tubes with 1 mL of Solution SR4. Invert at least once to mix and incubate at -20 °C for a minimum of 10 minutes.

*Start capping and labelling 1.5 mL sterile RNase free tubes (from zip-locked Axygen packets). For each sample, label two tube briefly for DNase treatment and then 4 extra tubes for aliquoting final RNA product, i.e., a total of 6 tubes per sample.

  1. Centrifuge the 2.2 mL Collection Tube at 13,000 x g for 15 minutes at room temperature to pellet the RNA.

*In the last 5 minutes of centrifugation, UV sterilise KimWipes in the BSC.

  1. Decant the supernatant and invert the 2.2 mL Collection Tube onto a paper towel for 10 minutes to air dry the pellet.

*Take TurboDNase buffer out of freezer to defrost. Prepare esky of ice.

  1. Resuspend the RNA pellet in 40 μL of Solution SR7 and place samples on ice.

TurboDNase treatment for 12 RNA samples

*Remove TurboDNase from freezer and thaw on ice.

*TurboDNase, buffer and DNase inactivation reagent should all be kept on ice while working with them and in the dark (covered esky).

  1. Using one of the 1.5 mL sterile tubes, make a DNase mastermix:
  2. FOR 12 SAMPLES: To 52 μL of TurboDNase buffer, gently add 13 μL of TurboDNase and very gently mix with your pipette tip.
  3. Transfer 5 µL of this DNase mastermix to each tube (there should be a minimum of 5 µL in each tube).
  4. Incubate samples for 20 min at 37 °C

*Take DNase inactivation reagent out of freezer to thaw.

  1. Add 4.5 µL of resuspended DNase Inactivation Reagent and mix well.
  2. Incubate 5 min at room temperature, mixing occasionally. Flick the tube 2–3 times during the incubation period to redisperse the DNase Inactivation Reagent.

*If room temperature cools below 22–26 °C, move the tubes to a heat block or oven to control the temperature. Cold environments can reduce the inactivation of the TurboDNase, leaving residual DNase in the RNA sample.

  1. Centrifuge at 10,000 × g for 1.5 min and transfer the RNA to an RNase free PCR plate.

*Avoid pipetting/disturbing the DNase Inactivation Reagent, it is difficult to remove, even with the magnet clean and interferes with downstream applications.

RNA Clean

  1. In PCR plate, pipette from aliquots 100 µL of RNAclean solution. Gently shake before to resuspend beads.
  2. Add ~100 μL sample and pipette to mix. Mix gently as fast pipetting can displace sample from well.
  3. Leave to stand for a minimum of 10 min to allow strands to bind to magnet beads.
  4. Transfer PCR plate to SuperMagnet. Leave for 10 min for rings to form.
  5. Carefully remove solution in wells without disturbing the ring of beads.
  6. Add 200 µL 70 % ethanol and leave for 30 s.
  7. Careful not to disturb the bead ring, pipette mix the ethanol to wash and remove from sample. Repeat for a total of 3 washes.
  8. After the last wash, make sure all ethanol is pipetted out from wells.
  9. Allow to air dry for 9-10 min. Do NOT allow beads to dry!
  10. Remove plate from magnet. Add 40 μL of DEPC RNase free water for RNA samples and pipette to elute beads and strands. Elution is rapid.
  11. Place plate back on magnet for 10 min.
  12. Carefully pipette out the sample into 1.5mL sterile tubes labelled earlier and place on ice.
  13. If you suspect carryover of magnet beads, place tube back on magnet plate and begin aliquoting 10 µL RNA to 3 x 1.5 mL tubes. With care, a final drop of sample containing magnet beads can be separated and removed.
  14. Place 3 x 10 µL RNA aliquots into -80 °C freezer. Keep 1 x 10 µL “dirty” sample on ice to nanodrop.
  15. The final RNA should be labelled according to the instruction given in the spreadsheet “sampling_schedule” and stored at -80 °C.