Chapter 3 Sample homogeneisation and digestion

Homogenisation (mechanically lyse) and chemical digestion of biological samples (including faeces, intestinal content, tissues, blood, saliva, urine, etc.) are crucial steps in DNA extraction. These steps help break down the complex matrix of the sample, thus separating different compounds and molecules from each other. Faeces, for example, are composed of a heterogeneous mixture of materials, including undigested food, microbial cells, host cells, and waste products. This complex matrix can make it challenging to access and extract DNA. Homogenisation involves breaking down the solid and semi-solid components of the sample, creating a more uniform mixture that is easier to work with, while digestion also entails degrading cellular structures to release intracellular compounds to the matrix.

The homogenisation/lysis step described below was added to the EHI laboratory workflow to aid the disruption process initiated at the time of sample collection in the field. To provide a reliable snapshot of the original microbiome profile, microbial cell lysis is first implemented by placing the sample in a buffer that serves a dual purpose: lysis and preservation. The buffer preserves genetic integrity and expression profiles at ambient temperatures, as it facilitates the release of nucleic acids from cells and tissues by breaking down cellular membranes and structures and simultaneously inactivates/denatures enzymes that can degrade nucleic acids.

The mechanical homogenisation step in the laboratory consists of agitating (bead-beating) samples with a lysing matrix, sometimes called grinding media or beads, in a bead beater. The EHI protocol uses a combination of ceramic, silica and glass beads for sample homogenisation. The mechanical force generated by the beads’ movement and collision with the sample causes physical disruption, breaking apart cells and releasing their contents into the surrounding solution. Optimisation of bead-beating conditions is necessary to balance efficient disruption with minimal degradation, as depending on the sample, bead types, and shearing time, there is a potential risk of shearing or damaging sensitive biomolecules.

However, due to the hardness, complete physical disruption of tissue samples could not be achieved even after mechanical homogenisation with Lysing Matrix D (MP Biomedicals). Therefore, we switched to an additional chemical digestion before DNA extraction.

3.1 Instruments, plasticware and reagents

Instruments

  • Bead mill/beater homogeniser e.g. TissueLyser II (Qiagen)
  • Analytical balance (for tissue sample)
  • Block Heater-Shaker e.g. ThermoMixer C (Eppendorf) (for tissue sample)
  • SAFE® Screw Cap De-/Capper (LVL technologies)
  • SAFE® 2D/1D Code Reader (LVL technologies)

Plasticware

Item Brand Catalogue number
Lysing Matrix E 96-Well Plate MP Biomedicals 116984001-CF
Polystyrene Tweezer (Single packed disposable sterile) Megro
Carbon steel surgical scalpel blades (Single packed sterile) 1C-X10-BL-CW-B-S
LX 1000 - 2D Biobanking Tubes LVL technologies 1C-X10-BL-CW-B-S

Reagents

Reagent | Brand | Catalogue number | Storage |
UltraPure™ 1M Tris Hydrochloride, Tris-HCI | Invitrogen | 15568025 | 4 ºC |
UltraPure™ 0.5M Ethylenediaminetetraacetic acid, EDTA | Invitrogen | 15575-038 | 25 ºC |
BioUltra Sodium dodecyl sulfate solution, SDS | Sigma Aldrich | 71736 | 25 ºC |
Proteinase K 20 mg/ml | Roche | 3115828001 | 4 ºC |

3.2 Protocol

Faeces and swabs

  1. Thaw the samples and ensure samples have entirely thawed.
  2. Spin down or gently centrifuge tubes briefly to remove any liquid from the lid.
  3. Add the content of 1 well of the Lysing Matrix E 96-Well Plate to each collection tube.
  4. Vortex to ensure that the beads are moving in each sample. If they are not moving despite vortexing, it indicates that the ratio between the sample and preservation buffer is not ideal (the amount of faecal material exceeded the 1:10 ratio). Consider using a sterile pipet tip to attempt to unclog the beads.
  5. Homogenise the sample with the TissueLyser for two rounds of 6 minutes at max speed (=30 Hz). Optimal settings should be optimised to ensure proper homogenisation with other bead mill/beater homogeniser instruments.
  6. Spin down or centrifuge the tubes at 2,2 g/rcf for 1 minute. Ensure no foam is present on the tube’s lids. Otherwise, repeat centrifugation.
  7. Gently transfer the supernatant to an LX 1000 LVL tube without disturbing the pellet and the beads for storage at -20ºC or -80ºC. We use LX 1000 - 2D Biobanking Tubes for space-saving storage and handling/processing of large quantities of samples. These tubes minimise the risk of cross-contamination, allow automatic traceability using the 2D code, and allow easy sample selection/reshuffling for processing. Moreover, they are compatible with automated laboratory systems.

Tissue samples

  1. Extract the tissue from the original preservation tube and weight it on a sterile weighing boat.
  2. If the tissue sample is heavier than 10 mg, cut a portion using a sterile scalpel and place it in an Eppendorf tube. Return the rest of the sample to the original preservation tube.
  3. Add 250 μl of SDS lysis buffer solution (Tris-HCL 0.1 M, EDTA 0.05 M, SDS 1.25%) and 25 μl of proteinase K to the tube. Vortex and spin down or gently centrifuge tubes briefly.
  4. Incubate overnight at 56°C on a a thermoshaker.
    1. Spin down or centrifuge the tubes at 2,2 g/rcf for 1 minute. Proceed to DNA extraction using 200 µl directly after lysis.