Sample Preparation

Scientific Support
Sample Preparation
Sample Preparation
General Sample Guidelines for Metabolon Studies
Guidelines for All Samples:
• Consistency in sample handling is very important. When collecting, it is important to
minimize operational variation (e.g. collection technique, time of sampling, time to
freezer, etc.). Materials collected for other experimental work and stored at -80°C can be
used for metabolomic studies as long as all of the samples were treated in a consistent way
during the collection process.
• Please notify Metabolon of any preservatives that have been used in the collection process.
• Please use polypropylene tubes for sending samples.
• Please use permanent markers (e.g. Sharpie) to directly label tubes while warm; cold
tubes will not hold the ink. Also, stick-on labels tend to fall off when frozen, so unless
specialized freezer-safe labels are available, direct marking is preferred.
• Samples should be shipped on dry ice. Tubes should be clearly labeled with sample
identifiers and include a hard copy of the list of samples to accompany the shipment.
Please include all available sample information when shipping samples, excluding any
patient identifiers in the case of human samples.
• Please send an electronic document containing sample ID information—preferably in
advance of sending the samples. Send to [email protected]. Send actual
samples to: Metabolon Sample Acceptance
617 Davis Drive, Suite 400
Durham, NC 27713 US
Phone: (919) 572-1711
Blood Samples:
1. a) Plasma: For human or other large-volume collection, collect whole blood in tubes (e.g.
Vacutainer or Vacuette) containing EDTA (K2, K3 or Na) as anti-coagulant. It is necessary
to mix the tube well after collection in the vacutainer, prior to centrifugation. After mixing,
centrifuge the samples at ~ 1,000 x g for 10-15 minutes. The clear, upper layer is plasma.
For small volume collection, such as small animal tail bleeds, keep the tube cold and quickly
determine the volume of blood, then add an equal volume of 9mM EDTA in PBS (to achieve
a final concentration of EDTA of approximately 4.5 mM). Mix well and keep cold until
centrifugation. As a general guideline, 1.8 ml of whole blood will yield ~1 mL of plasma.
Plasma Anticoagulant Guidelines:
• Best Results: EDTA (K2, K3, Na; avoid Li)
• Acceptable Results: Citrate
• Less Desirable: Heparin
• Absolutely avoid including multiple anticoagulant sample types in the same
experiment (e.g. EDTA plasma, heparin plasma, serum).
b)Serum: Collect whole blood in serum separator tubes and follow tube manufacturer’s
processing instructions.
c) Whole blood: Whole blood must be prevented from coagulating, preferably by the
addition of EDTA. See the plasma preparation guidelines above, but freeze the sample
immediately after adding the anti-coagulant and mixing well.
2. Volume: 100-200μl is preferable with 200μl providing a potential backup sample if
needed. It is also preferable to have consistent sample amounts for all samples. Avoid
having samples sit at room temperature for more than 15 minutes if possible, but noting
that consistency of handling is most important.
These guidelines are provided to help
Metabolon clients understand typical
sample types and amounts for analysis.
Please talk to your Metabolon
contact about specific questions with
regards to your particular experiment
and sample amounts & types.
3. Immediately aliquot the resulting plasma/serum/whole
blood into chilled, polypropylene tubes. We prefer the
following: Sarstedt tubes with O-ring seal, 2 mL (Sarstedt
Cat # 72.694.006 or Fisher Cat # 50809242). Eppendorf
(Brinkmann), 1.5 mL polypropylene may also be used
(Brinkmann Cat # 022363204 or Fisher Cat # 05-402-25).
4. Immediately freeze the tubes containing the plasma/serum/
whole blood. Flash freezing in liquid nitrogen is ideal.
However, if these resources are not available, immediate
placement of tubes in a -80°C freezer or dry ice/ethanol bath
is acceptable (NOTE: ethanol may wash off labeling!).
Storage should remain at -80°C until shipment.
Urine Samples:
1. Collect 100-200 μl of urine sample in sample vials as
described above (step 3 in Blood section). 3. Ideally, each vial sent to Metabolon contains the same number
of viable cells, but in any case the cell counts for each vial
should be defined. For non-attached cells, go to step 5.
4. For attached cells, trypsinize or scrape cells according to
established conditions in your laboratory. Gentle trypsinization
may result in a more reproducible yield of cells and reduced
cell lysis.
5. Transfer the cell suspension to 15 mL polypropylene tubes
(BD-Falcon Cat # 352097 or Fisher Cat # 14-959-70C) or,
if a larger volume is needed, a 50 mL polypropylene Falcon
Tube may be used (Falcon Cat # 352070 or Fisher Cat #
14-432-22).
6. Spin at about 750-1000 xg for 1-3 minutes to pellet cells, or use other conditions that have been optimized in your
laboratory. Avoid spinning conditions that will lyse the cells.
Remove and discard all but ~0.75 mL of the supernatant.
2. Immediately after collection, freeze sample as quickly as
possible and store at -80°C.
Tissue Samples:
1. For collected solid tissues (e.g. biopsy material), the amount
of tissue/sample can vary depending upon study objectives
and tissue type—typically, a 50 mg sample will suffice.
However, the exact weight of tissue collected is best determined
for each study on a case-by-case analysis during design
discussions with Metabolon scientists.
2. Metabolon has experience with as little as 10-50 mg of tissue.
Please contact your Metabolon representative with questions.
3. Transfer sample to cryovials, 2.0 mL polypropylene. (Fisher
Cat # 5011-0020 or Fisher Cat # 03-341-18D/Wheaton Cat
# 985734).
4. Flash-freeze sample (as described above) and store at -80°C
until shipped.
5. When possible, the material should be shipped in tared vials.
6. Tissues frozen in the presence of OCT is acceptable, but not
preferred. In any case, all samples in a study should be
similar in this respect.
Cell Culture Samples (Eukaryotic and Bacterial):
1. In general, a 50 μl packed pellet of cells is acceptable and a
100 μl packed cell pellet preferable for analysis.
2. Cultures should be prepared with cells grown under various
conditions as defined in the study design. (Note that if the
study conditions lead to large differences in cell volume
between treatment groups, this may affect results). Harvest
the cells and prepare for shipment to Metabolon as described
Corporate Headquarters
Research Triangle Park, North Carolina
+1.919.572.1711
below. Optionally, also save 200 μl cell media [spin to pellet
residual cells and debris and collect top layer (supernatant)]
during harvesting for metabolomic analysis.
7. Gently resuspend cells and transfer to pre-labeled cryovials,
2.0 mL polypropylene. (Fisher Cat # 5011-0020 or Fisher
Cat # 03-341-18D/Wheaton Cat # 985734).
8. Spin (750-1000 x g for 1-3 minutes) again and carefully
remove all supernatant. If possible, use narrow-bore (gelloading or small volume) micropipette tips to minimize
buffer residual and ensure a dry pellet for freezing.
9. Flash-freeze sample (as described above) and store at -80°C
until shipped.
Plant Samples:
1. Fresh plant material should be harvested into liquid nitrogen.
2. Grind the sample to a powder in mortar and pestle under
liquid nitrogen. Preferably, the powdered material should be
lyophilized. Transfer the powder to a polypropylene tube for
shipping.
3. If needed, sterile sand can be used to aid grinding, but an
accurate measure of plant material and sand in the mixture
is necessary to standardize sample size.
4. Each powdered sample should contain approximately 50 mg
or more (dry weight), or 100 mg or more (fresh weight) of
plant material.
5. Ship on dry ice.
For other sample types or questions please consult
with Metabolon scientists.
European Headquarters
London, England
+44 (0) 20 3 318 5807
Madrid, Spain
+34 (0) 609 106 782
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www.metabolon.com
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