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RNA Extraction Microbiome - Fecal

Theory

This protocol is for the extraction of RNA from fecal samples including fresh fecal samples, frozen fecal samples, liquid fecal samples, and fecal swabs.

Prepare Your Workspace

You will be doing the DNA plate and sample preparation in the biological safety cabinet to keep the environment around you as sterile as possible. Extraction will be done by the KingFisher Flex in the lab.

  • Turn on the Biological Safety Cabinet. This is done by lifting the sash to operating sash height, marked on the left side of the hood. This should turn on the blower, white light, and window alarm.
  • Clean the cabinet with 70% ethanol, including the work surface, walls, and glass.
  • Lower the sash and press the UV button. This will turn on the UV light for 15 minutes. It will turn off automatically.

⚠️ You cannot expect the glass to protect you from UV exposure. You can be in a different part of the lab while the light is running, but do not loiter in front of the cabinet.

Remember, before placing any objects in the hood, they must be sprayed with 70% ethanol.

Gather Materials

  • MagMAX™ MICROBIOME Ultra Nucleic Acid Isolation Kit (Thermofisher Scientific, A42358)
  • 200PF Molecular Grade Ethanol (VWR, 71006-012)
  • Nuclease-Free Water
  • Sterile Pipette Tips (P100, P1000)
  • Serological Pipettor
  • Serological Pipettes (25, 50)
  • 50 mL conical tube(s)
  • Plate Tube rack
  • Reagent Reservoirs
  • 5 Deep-Well Plates (ThermoFisher Scientific, 95040460)
  • 2 Elution Plate (ThermoFisher Scientific, 97002540)
  • 1 Tip comb (ThermoFisher Scientific, 97002820)
  • Plate Films
  • Bead tubes (ThermoFisher Scientific, A42351)
  • Zymo Microbial Community (VWR, 77001-842)
  • VWR marker
  • Scissors
  • Plate Map (reference “2.3_Plate-Map-Template”)

Protocol

Lyse Sample

  1. Add 800uL of Lysis Buffer to bead tubes.
  2. Prepare fecal samples as follows:

Table 1. Fecal sample preparation.

Sample Type Amount to Add
Fresh/Frozen Fecal Samples Weigh out 100mg, then place into prepared bead tube
Fecal sample in storage solution Remove 200uL, then place into prepared bead tube
Fecal Swab Remove the plastic/wooden stick, then place swab into prepared bead tube
  1. Place tubes in bead ruptor for 10 minutes at 20 Hz.
  2. Remove tubes from bead ruptor and centrifuge at 12,500rpm at 25°C for 2:00 minutes. At this point, bead tubes can be stored at 4°C overnight or -20°C for up to 3 months.

Prepare Plates

Prepare the 5 plates as follows:

Table 2. Plate types, reagents, and volumes.

PLATE ID PLATE TYPE REAGENT VOLUME PER WELL
Tip Comb Standard Plate Place a 96 deep-well tip comb on a standard plate
Sample Plate Deep Well Sample + Binding/Bead Mix
Wash 1 Deep Well Wash Buffer 1000uL
Wash 2 Deep Well Wash Buffer 1000uL
Wash 3 Deep Well 80% Ethanol 1000uL
Wash 4 Deep Well 80% Ethanol 1000uL
Elution Standard Well Elution Buffer 200uL*

*can be reduced to 100uL if 1 fecal swab

  1. Prepare mixture of Viral/Pathogen Binding Solution + DNA/RNA Binding Beads Combine 500uL/sample of Viral/Pathogen Binding Solution and 20uL/sample of DNA/RNA Binding Beads. ❗Theses solutions are very viscous ❗

Table 3. Bead Binding Mix volumes. For a full plate make this reaction in each of two 50mL conical tubes.

Solution Volume
Viral/Pathogen Binding Solution 25mL
DNA/RNA Binding Beads (vortex vigorously first) 1mL (or 1000uL)
  1. Prepare 80% Ethanol Wash Buffer. Make enough 80% Ethanol for 2mL/sample. Check the shelf with the kit reagents for leftover 80% Ethanol in a flask.

Table 4. 80% Ethanol Volume. Make in a clean flask for a full plate.

Solution Volume
100% molecular grade ethanol (in flammable cabinet) 160mL
Invitrogen UltraPure Water 40mL
  1. Prepare Sample Plate
  2. Transfer 400uL of sample to appropriate well of a deep-well sample plate.
  3. Invert Binding Bead Mix and add 520uL to each sample in the sample plate. ⚠️ This solution is very viscous. Use a fresh pipette tip for each well.
  4. Proceed directly to the KingFisher Flex.

KingFisher Processing

  1. Turn on the KingFisher Flex (the on switch on the bottom left of the machine).
  2. Push the right arrow to select "User" (the middle tab), then press the down arrow to select "DNA."
  3. Push the down arrow until you find "MagMAX_Microbiome_Stool_Flex" and then press "Start."
  4. Load your prepared plates onto the machine following the prompts on the screen. Be sure to remove the plate seal. Press the "Start" button to advance to the next plate. ❗ Be sure that the plate is oriented correctly, with the A1 corner of the plate matching the A1 label on the KingFisher plate dock.
  5. Once all plates are loaded, press "Start" to begin the protocol.
  6. After the program completes, you will be prompted to unload your plates. Keep the elution plate, seal with fresh plate film and store at 4°C. If storing for longer term, transfer elution to nonstick DNA tubes and store in the -80°C freezer.
  7. All other plates can be discarded in the biohazard waste bin.
  8. Turn off the Kingfisher and wipe down with 70% ethanol.

DNA Removal

  1. Measure concentration of RNA and DNA on Nanodrop.
  2. The treatment you use depends on how much DNA contamination you have. a. Routine (<200ug of nucleic acid per mL [< 200ng/uL]) i. Add 0.1 volume 10X DNase I Buffer. ii. Add 1uL rDNase I. iii. Mix gently. b. Rigorous (>200ug of nucleic acid per mL [> 200ng/uL]) i. Dilute sample to 200ng/uL a. Add 0.1 volume 10X DNase I Buffer. b. Add 0.5uL of rDNAse I, incubate for 30 minutes at 37C. c. Add another 0.5uL of rDNase I, incubate for 30 minutes at 37C. ii. If sample cannot be diluted a. Add 0.1 volume of 10X DNase I Buffer. b. Add 2-3uL of rDNase I.
  3. Incubate at 37C for 20 - 30 minutes. a. If you diluted, skip this step.
  4. Add 0.1 volume resuspended DNase Inactivation Reagent, mix. (For Routine, use 2uL or 0.1 volume, whichever is greater.) (For Rigorous, add 0.2 volumes if not diluted.)
  5. Incubate at room temperature for 2 minutes. Mix occasionally.
  6. Centrifuge at 10,000 x g for 1.5 minutes. (For 96-well plates, centrifuge at 2,000 x g for 5 minutes.)
  7. Transfer supernatant to fresh tube. This is your RNA and is very unstable.