Contact Kathryn Lesko (Technician for Dr. Jabri) with questions:
klesko@medicine.bsd.uchicago.edu
SHARE Biospecimen Protocol: Stool Sample
Supplies:
Stool Collection frame and bowl: Medical Action Industries Inc. cat# 02072
Fecal Collection tubes: Sarstedt cat#80.623
TE buffer: 30 mM Tris·Cl, 1 mM EDTA, pH 8.0
Lysozyme: Sigma-Aldrich cat # L6876
Proteinase K: Qiagen cat # 19131
RLT Plus Buffer: Qiagen cat #1053393
Beta-Mercaptoethanol (BME): Sigma-Aldrich cat # M6250
Bead beating tubes with 0.5mm Glass Beads: MoBio cat # 13116-50
MoBio PowerMag® Microbiome RNA/DNA Isolation Kit: MoBio cat # 27500-4-EP
Stool Sample Collection Instructions
1) Place stool collection frame on the back of the toilet bowl under the toilet seat. All
four corners of the collection frame should be supported by the toilet bowl. Place
collection bowl in the frame.
2) Place the toilet seat down. Do Not urinate into the collection container. Deposit
stool directly into the collection container.
3) After collecting sample, remove container from the frame.
4) *Unscrew the short, wide tube and use the scoop on the lid to transport one large
scoop from the white collection bowl into the tube. Screw down the cap tightly.
5) Unscrew the tall, thin tube and transport one large scoop into this tube. Screw
down the cap tightly.
6) There will be three stool samples from the collection:
a. Stool sample in RNAlater (for DNA and RNA isolation)
b. Stool sample in ethanol (for metabolite and DNA analysis)
c. Stool sample with nothing added (for DNA and metabolite analysis)
Stool Sample Processing Instructions
a. The ethanol sample will be stored at -80C.
b. The sample with RNA later will be stored at 4ºC for 24-48h . Then
remove RNAlater and freeze biopsies at -80º.
c. Stool sample with nothing added: process day of the collection, at
room temperature.
1. Add 110 ul of TE buffer containing lysozyme and proteinase K to
the stool pellet
- TE buffer: (30 mM Tris·Cl, 1 mM EDTA, pH 8.0) containing
15 mg/ml lysozyme.
- Add 15-20ul of Proteinase K into 200ul of the TE buffer
containing lysozyme (15mg/ml) for each sample.
2. Mix well by vortexing for 10 sec.
Note: If sample is frozen, add enzymatic lysis buffer directly to frozen sample
without thawing.
3. Mix well by vortexing for 10 sec.
Contact Kathryn Lesko (Technician for Dr. Jabri) with questions:
klesko@medicine.bsd.uchicago.edu
4. Incubate at room temperature (15–25°C) for 10 min. Incubate on a
shaker or vortex for 10 sec at least every 2 min.
5. Add 1200 ul of RLT Plus with BME (1:100 BME to RLT Plus) to
sample tube and mix well by vortexing for 10 sec.
6. Transfer sample into a 2 ml sterile bead beating tube filled with 1
ml of 0.1mm glass beads.
7. Fill the remaining volume of tube with RLT Plus w/ BME before
adding the screw top cap.
8. Place bead tube in bead beater for 3 min on “homogenize” setting.
9. Remove the tube from the bead beater and centrifuge for 5 min at
max speed to pellet debris.
10. Transfer the supernatant from bead tube into a sterile 1.5ml tube.
11. Centrifuge for 2 min at max speed to remove any additional debris.
12. Proceed with the MoBio PowerMag® Microbiome RNA/DNA
Isolation Kit.
*Note: This is an idealized protocol. If patients are unable or unwilling to move
stools to appropriate tubes, full sample should be sent to lab for processing, sameday if possible.