Metabolomics Core Facility Sample Preparation Guidelines General recommendations for metabolomics analysis: sample preparation, handling and precautions General technical recommendations: -‐ -‐ Use permanent markers to label your samples and add clear tape to protect them from organic solvents and extreme cold. Ensure that the labels are legible. Please, provide as much information as possible to help us before sending your samples. Once we have received and reviewed your request, we will send you an Excel spreadsheet by e-‐mail to be filled with your sample information (sample ID, etc…) as well as a cost estimate for your analysis. We will also send you a sample extraction protocol that has been optimized for the extraction of the metabolites you have requested in the sample submission form. If there are any questions we will contact you directly by e-‐ mail. -‐ Ship samples to: Dr. Daina Avizonis Metabolomics Core Facility, Goodman Cancer Research Centre 1160, Pine Avenue W, Room 401A, H3A 1A3 Montreal, QC, Canada e-‐mail: [email protected] Telephone : 514-‐398-‐5199 -‐ -‐ -‐ -‐ Please make sure there is enough dry ice in your package to prevent any sample thawing or degradation. Avoid shipping on Thursdays or Fridays since samples might not be received before the week end. A hard copy of the list of samples sent should be printed and included in the shipment package. Please include all available sample information, excluding any patient identifiers. We provide buffers compatible with mass spectrometry analysis and high quality grade solvents for washing and extraction, please do not hesitate to ask for solvents or product reference. Please note that we do not process biohazardous or radioactive samples. General handling recommendations: A pilot study is highly recommended. One test extraction on each cell or tissue type is strongly advised as a small pilot study to determine if this extraction technique is clear and will work for your particular cell type/tissue and to determine the correct number of cells or tissue weight to give the best results. -‐ -‐ -‐ -‐ -‐ -‐ -‐ Any additive, detergent or preservative must be avoided in your sample, please inform us if your samples require any additives or stabilizers before proceeding with any sample preparation. Some additives are incompatible with mass spectrometry. Work as fast as possible and under cold conditions to prevent any possible further metabolism variation. Metabolic changes will continue in any fluid, tissue or cell with active enzymes. Metabolite levels can change rapidly, measures must be taken to stop metabolism so that a metabolic “snapshot” could be taken and groups compared (e.g. KO vs WT). Be sure to grow, harvest and sacrifice under the same conditions. It is always better to process all your samples the same way, at the same time and using the same set of reagents. Try to be consistent/reproducible with each and every step. Prepare everything you need in advance: chilled solvents, labelled tubes, ice and bucket, dry ice and bucket, liquid nitrogen... Keep your samples as cold as possible all the time to prevent degradation. Please, do not add or remove steps from the protocol provided, ask us if you have any question. Metabolomics Core Facility Sample Preparation Guidelines 1) Intracellular metabolomics analysis: -‐ -‐ -‐ -‐ -‐ -‐ -‐ -‐ -‐ -‐ -‐ Metabolomic studies on cells can be done on 6 cm, 10 cm or 15 cm plates depending on your cell size and growing conditions. We do not recommend multi-‐well plates since they cannot be quenched and processed individually. Usually, 1-‐10 million cells are required for metabolomic analysis depending on the analysis and natural abundance of the metabolites of interest. Please note, the cell size may influence the amount of material needed to get an acceptable signal (do a pilot study). Provide the MCF facility with the number of cells per plate, and the amount of protein per plate so that we can prepare the samples for analysis accordingly. The time that the media is changed should be noted and 1.5 ml of media should be kept frozen if analysis of metabolite uptake and excretion is desired (different protocol). If duration of treatment is critical, please stagger the start times to allow time to perform the manipulation (wash/quench) for each plate. Check under a microscope to see if cells are disrupted or lysed by the wash solvent used in the protocol. Be sure that tissue cultured cells are free of mycoplasma contamination. Remove one plate at a time from the incubator and process immediately. Please, note that we do not perform cell count or protein amount determination on individual samples. At least one parallel plate for cell counting of each cell condition should be grown in parallel for metabolomics analysis (3 plates are recommended to get an average cell count or protein amount). The same plate should be used to measure protein content. Cells of each population that are to be analyzed by the MCF should be grown under the same conditions as much as possible (media changes at same time, same cell density, etc). Cell counting and/or protein amount determination should be performed in your lab in advance for data normalization purpose. Cell counts and protein amounts should be recorded and sent along with the samples for analysis by the MCF. A minimum of 3-‐5 replicates (or more!) per cell condition are required for unbiased analysis and reasonable statistics depending on the analysis required. More replicates may be required for untargeted analyses. The use of a vaccum aspirator is recommended for washing steps to ensure efficient removal of all culture medium. Most laboratories do not have all of the necessary equipment to complete cell extractions. The extraction protocols have “stop points” indicated where the extraction can be stopped and samples sent to the facility where we will complete the extraction. If there are any questions, please do not hesitate to contact us. 2) Tissue metabolomics analysis: -‐ -‐ -‐ -‐ Generally 5 to 50mg of tissue are required for analysis. This is highly tissue and treatment dependent as well as dependent upon the specific metabolites to be measured. For tissue, organ or tumor analysis, cardiac puncture should be performed on the animal when possible as well as perfusion with cold saline solution (e.g. 150 mM ammonium formate solution pH 7.4) to remove blood from tissues. Tissue harvesting (e.g. surgical or biopsy material) should be performed rapidly using a freeze clamp technique or similar when possible to preserve sensitive metabolite levels. Samples should be frozen rapidly in liquid nitrogen followed by crushing in a liquid nitrogen cooled mortar. Powder should be weighed for normalization. Consistent weights across the different tissue conditions or treatments should be provided to ensure similar limits of sensitivity. Metabolomics Core Facility Sample Preparation Guidelines -‐ A minimum of 5-‐10 replicates (or more!) is recommended per tissue condition for unbiased analysis and reasonable statistics (higher variability compared to tissue culture). More replicates may be required for untargeted analyses. Most laboratories do not have all of the necessary equipment to complete tissue extractions. The extraction protocols have “stop points” indicated where the extraction can be stopped and samples sent to the facility where we will complete the extraction. If there are any questions, please do not hesitate to contact us.