All projects require submission of a request for project (RFP) application through iLab and approval for relevance and scientific merit. Before initiating the project and submitting samples, a meeting is required with principle Investigator requesting the service and core directors and biostatisticians for design of the project. At that time, investigators will receive additional information on sample preparation and scheduling of the project. At the completion of the project another exit meeting with the Principle Investigator is required to receive the report and explanation of the tier 1 data analysis. Data analysis will not be released without this meeting.
All users of the core are required to sign up on iLab for requesting services and provide account sources for core charges. Sample submission and preparation for analysis by the core must be accompanied by a completed sample submission form. Baylor investigators may complete and submit the form online (iLab) or by email electronic copy and below to core director.
Nagireddy Putluri (email: Putluri@bcm.edu)
Rules and Requirement for Collection and Submission of Samples
Guidelines for all samples:
1. Please notify us of any preservatives or additives that have been applied during the collection process.
2. Use permanent markers (e.g. Sharpie) to directly label tubes since labels may fall off when frozen.
3. Send an electronic document containing sample ID information—preferably in advance of sending the samples. Send the completed xl file to core directors.
4. All Samples should be frozen and on dry ice. Tubes should be clearly labeled with sample identifiers and include a hard copy of the list of samples. Please include all available sample information when you submit the samples, excluding any patient identifiers.
Cell line samples:
1. Prepare the 13C labeled growth medium with the recommended concentrations. Nutrients labeled with 13C must be purchased from Cambridge Isotope Laboratories. Cells are grown in regular medium until 80% confluence, followed by overnight starvation in metabolite free medium and then addition of tracers L[U-13C5]glutamine, [1-13C]glutamine, or [5-13C]glutamine supplemented with medium containing [-]glutamine with 10% dialyzed FBS and 1% PS, or addition of D[U-13C6]glucose supplemented with media [-]glucose, 10% dialyzed FBS, and 1% PS. Culture medium is collected, cells are washed with PBS, and equal numbers of cells from each treatment are snap-frozen with liquid nitrogen. Cells are scraped into a 0.5-ml mixture of 1:1 water/methanol. (Note: Depends on the medium the concentration of L[U-13C5]glutamine or [U-13C6]glucose will change).
2. Provide 500 ul of cell lysates to the core
3. A minimum of 3-5 million cells/pellet is required. We recommend 4-5 biological replicates for each experimental group.
Investigators are required to submit the test samples (at least 3 samples) to check the labeling efficiency at different time point. For this there will additional change and need to discuss with core directors.
Additional details on cell cultures and culture conditions are required. This is critical as it can effect detection of metabolites. This information must be provided on sample submission form.
1. For permanent cell lines provide cell line name. For primary cell cultures whether it is human patient derived.
2. Provide detailed and accurate information on culture conditions and treatments.
3. Culture medium, serum and other supplements:
4. Provide information on any drug treatment of cell cultures: Drug name, dosage, and chemical structure. Drugs can potentially interfere with our analysis.
5. Knockdown treatment of cells. (si or shRNA). Documentation of knockdown efficiency is required (protein data).
Sample submission and Q/C process
500 ul of above mentioned lysate will use further processing and mass spectrometry analysis performed. If sample amounts did not meet minimum criteria ( ie number of cells) or quality ( variation in cell numbers between samples) the Core reserves the right to reject samples for further analysis. The core will consider analysis of rejected samples in cases when they are irreplaceable or too costly to replace. Under these circumstances analysis can only proceed with the understanding and acknowledgment by the principle investigator of the risk of failure to obtain quality results and an agreement to pay core charges regardless of the results.
A determination of optimal time points is required for each project. Preliminary samples for a pilot MS flux analysis is required in advance of analysis of the entire sample set to determine Q/C and to fine-tune parameters. If the pilot analysis fails to detect metabolites accurately the core will not proceed with the entire set of experimental sample. After pilot samples have passed Q/C the Core will proceed with analysis of the experimental samples.
All samples of an approved project must be collected before submission to the core and for analysis. The core will not process partial samples from an experimental protocol. Samples will only be processed all together. After receiving samples that pass Q/C Core personnel will perform all procedures including extractions of samples for different class of metabolites, column chromatography for each class of metabolites, mass spectrometry analysis, Q/C and normalization of mass spectra and tier 1 analysis of data.