Guidelines for Metabolomics Sample Collection

Metabolomics Recommends Sample Size

Animal Tissue

Processing Steps:

1. Animal tissue samples: If collecting the entire tissue, use perfusion method with pre-cooled deionized water to remove residual blood. If collecting partial tissue, rinse off residual blood with pre-cooled deionized water after tissue disruption.
2. Human tissue samples: For small biopsy samples, quickly rinse with pre-cooled deionized water to remove residual blood.
3. Take specific portions according to the experimental design and place 250 mg/sample into centrifuge tubes.
4. After labeling, promptly place the tubes in liquid nitrogen for at least 15 minutes for cryopreservation.
5. Store at -80°C freezer. Sufficient dry ice should be used during shipment.

Notes:

• Strive to keep the sampling sites consistent for biological replicates.
• Take precautions to avoid contamination from anesthesia, Eppendorf tubes, collection instruments, etc., during tissue sample collection and processing.
• Sample collection should be done quickly and on ice to minimize the storage time at -20°C or -80°C freezers.
• Divide the samples promptly to avoid repeated freeze-thaw cycles.

Liquid Samples

Serum Samples

Collect blood in centrifuge tubes or vacuum blood collection tubes and let it clot for 1 hour at 37°C (or room temperature). Then, centrifuge at 3000 rpm for 5 minutes, transfer the supernatant to clean centrifuge tubes. Centrifuge again at 12000 rpm for 10 minutes at 4°C, transfer the supernatant to 2 mL centrifuge tubes, 0.2 mL per tube. After labeling, flash freeze in liquid nitrogen for 15 minutes, and store at -80°C in the freezer. Sufficient dry ice should be used during shipment.

Plasma Samples

Recommend using sodium heparin anticoagulant tubes to collect whole blood and separate plasma as soon as possible. Centrifuge at 3000 rpm for 10 minutes at 4°C, collect the upper layer, and distribute 0.2 mL per tube into 2 mL centrifuge tubes. After labeling, flash freeze in liquid nitrogen for 15 minutes, and store at -80°C in the freezer. Sufficient dry ice should be used during shipment.

Note:

• Animals should be fasted without water for at least 10 hours before sampling, and for clinical samples, a light diet should be followed with fasting and water restriction after 8 PM on the day before sampling. It is important to maintain consistency in the sample handling process, including sample placement time and centrifugation time.
• If alcohol is used for disinfection before blood collection, dry the area completely before sampling, ensuring that the alcohol has completely evaporated.
• Choice of blood collection tubes: For plasma, it is recommended to use sodium heparin anticoagulant tubes (green cap). Citrate anticoagulant tubes (blue or black cap) and EDTA anticoagulant tubes (purple cap) may interfere with metabolites and have matrix effects, so their use should be avoided as much as possible. For serum, use vacuum blood collection tubes without anticoagulants (red cap) during blood collection.
• It is recommended to collect as many samples as possible and distribute them into 2 mL centrifuge tubes for freezing and storage, avoiding repeated freeze-thaw cycles.
• Serum reference yield: 30%~50% (for example: 1 mL of whole blood can obtain about 0.3~0.5 mL of serum); Plasma reference yield: ≈50% (for example, 1 mL of whole blood can produce about 0.5 mL of plasma).

Ascites Fluid Samples

Collect ascites fluid in centrifuge tubes, flash freeze in liquid nitrogen, store at -80°C, and ship with sufficient dry ice.

Urine Samples

Collect midstream urine (clinical) or urine from the first hour of the morning (animals). Centrifuge at 3000 rpm for 10 minutes at 4°C, collect the clarified urine from the middle layer, and distribute 500 μL per tube into centrifuge tubes. After labeling, flash freeze in liquid nitrogen for 15 minutes, and store at -80°C in the freezer. Sufficient dry ice should be used during shipment.

Fluid Samples (e.g., joint fluid, saliva, bile, cerebrospinal fluid, rumen fluid, etc.)

1. Collect the fluid samples.
2. Centrifuge at 3000 rpm for 15 minutes at 4°C, collect the supernatant, and distribute 0.5 mL per tube into 1.5 mL centrifuge tubes.
3. Store at -80°C in the freezer. Sufficient dry ice should be used during shipment.

Cell Samples

Suspended Cells

Collect suspended cells by centrifugation, wash quickly 2-3 times with pre-chilled PBS, centrifuge at 4°C, 1000g for 1 minute, discard the supernatant, and collect the cells in a 2 mL cryotube. Flash freeze in liquid nitrogen for 15 minutes, store at -80°C, and transport with sufficient dry ice.

Adherent Cells

Remove the culture medium from the cultured cells, wash 2-3 times with pre-chilled PBS, discard the supernatant, and add 1 mL of pre-chilled 60% methanol-water solution (chromatography grade). Scrape off all cells from the culture vessel using a cell scraper, centrifuge at 4°C, 1000g for 1 minute, discard the supernatant, and collect the cells in a 2 mL cryotube. Flash freeze in liquid nitrogen for 15 minutes, store at -80°C, and transport with sufficient dry ice.

Fecal and Intestinal Content Samples

Processing Steps:

After collecting fresh fecal or intestinal content samples, divide the samples into 250 mg portions per tube. Immediately flash freeze the samples in liquid nitrogen for 15 minutes. Once properly labeled, store the samples at -80°C in a freezer and ship with an ample supply of dry ice.

Notes:

Due to the presence of a multitude of microorganisms in fecal/intestinal content, which have rapid metabolic activity, repeated freeze-thaw cycles can significantly impact the metabolic profile. It is recommended to divide and store the samples as 250 mg portions after collection.

Plant Tissue (Leaves, Stems, Flowers):

Take a whole leaf/segment of stem/flower, place it in a centrifuge tube or aluminum foil, and label it. Quickly freeze it in liquid nitrogen for at least 15 minutes.

Place the tubes containing the samples into self-sealing bags (one bag per group) and include a label inside the bag indicating the sample information.

Store the samples promptly at -80°C in a freezer and ship with an ample supply of dry ice.

Note: (1) When collecting leaf samples, it is preferable to collect them during the well-lit hours of noon. (2) Maintain consistency in sample collection, particularly within the same group of samples (color, degree of aging, proportion of veins, light exposure, position, etc.).

Roots Samples:

Take the roots of the whole plant and quickly rinse off any soil, culture medium, or nutrient solution with PBS buffer or ultrapure water.

After drying with absorbent paper, divide the roots into 500 mg portions per tube.

After labeling, promptly freeze them in liquid nitrogen for at least 15 minutes, store at -80°C, and ship with dry ice.

Fruits, Seeds Samples:

For fruits with high water content and larger volume (e.g., tomatoes, watermelons, apples), divide the samples into "uniform" small pieces (≈ 200 mg/sample) and place them in 2 mL centrifuge tubes. After labeling, quickly freeze them in liquid nitrogen for at least 15 minutes.

For small granular seeds (e.g., Arabidopsis seeds, cereal seeds), mix the seeds from the same group and then divide them (≈ 200 mg/sample) into centrifuge tubes. After labeling, promptly freeze them in liquid nitrogen for at least 15 minutes.

For whole fruits that require extraction (e.g., whole grapes), place the fruits in 50 mL centrifuge tubes/self-sealing bags. After labeling, promptly freeze them in liquid nitrogen for at least 15 minutes, store at -80°C, and ship with dry ice.

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