|Molecular Profiling (Genetics)|
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To Begin a Project and Schedule Service:
Before initiation of a study requiring collection of human or animal specimens, and testing by the Molecular Profiling Facility, schedule a consultation with Tapan Ganguly at firstname.lastname@example.org for specimen collection, storage, and delivery to the Facility. The Facility works closely with the Penn Bioinformatics Core, and new clients are strongly encouraged to schedule a bioinformatics consultation to cover experimental design, analysis strategies and tools. The principal investigator to be billed can create and manage projects by visiting How to Request Services. Remember to authorize all users for your project who will be allowed to Request Service and have direct access to your data. Complete the appropriate Service Request Form in the Service Section and submit it online, entering the assigned Project ID. Your online form will be reviewed, once approved you will receive an email. Please deliver your samples on dry ice to 68 John Morgan Bldg. between 1 pm-3 pm on Tuesdays and Thursdays or schedule a time with the Facility. Please bring a print out of the Request ID with your samples. The order of your samples in our log book determines your priority in the sample processing schedule. To request work by the Facility staff on your samples, follow Sample Submission Guidelines. To reserve self-service instruments, go to Order Training. Once you complete your training you can use Schedule Self- Serve Equipment.
Sample Submission Guidelines
Sample submissions require a Project ID. Principal investigators may view authorized projects, or create a new project, using the Manage Project IDs link in the left menu on this page. For TUBES, label every tube on top clearly, using exactly the same FACILITY ID as on your submission form. For sample PLATES, label every plate clearly on FRONT SIDE using the same FACILITY ID and REQUEST ID as on your submission form. Mark as SAMPLES. For assay PLATES, label every plate clearly on FRONT SIDE using the same REQUEST ID as on your submission form. Mark as ASSAYS. Assay list can be send by email and the orientation should match with the assay tubes/wells.
All human and animal specimens must be treated as potential infection risks. Care should be taken to avoid over-filling of blood tubes, which is likely to be associated with leakage of blood and contamination of the external surface of the container
All human and animal specimens should be transported to the laboratory in biohazard bags, with minimum delay. If delay is inevitable, it is generally better to refrigerate samples in the interim, however refrigeration may itself cause changes in the results. Samples should not be subject to temperatures of >25°C, even for short periods
A. For DNA Extractions
If the person taking blood, does not invert the tube several times before storing it or sending it off to a lab for analysis, then it will clot, even with the anticoagulant in there, as it has to actually be mixed in with the blood. Even if the draw is done properly and the vaccutainer is mixed properly, it is still possible for the blood to clot. Sometimes it is a result of a bad vaccutainer, poor dispersal of the additive within the tube, bad seal, or possibly from the venipuncture taking too long to fill the tube. After 1 week of storage at room temperature, blood samples collected in both the anticoagulants began to show signs of DNA degradation and reduced yield. High-molecular-weight DNA was purified from blood samples stored at 4°C for up to 1 week, but the DNA yields from these samples slightly decreased for both the anticoagulants. For DNA, frozen blood can be used. Transfer blood to cryogenic vials in aliquots labeling appropriately or a 15 ml conical tube depending on the size you want the DNA to be extracted. Freezing at -80°C should be done as early as possible. High-quality DNA can be purified from all blood samples stored at -20°C and -80°C for up to 4 weeks, but samples stored at -20°C resulted in lower DNA yields than those stored at -80°C.
Cheek swabs are a non-invasive method of collecting DNA. No food or drink should be allowed for twenty minutes prior to sampling.
B-Cells, T-Cells, Dendritic Cells, PBMCs or cell lines should be delivered in frozen pellet form
B. For RNA Extractions:
We extract from Paxgene blood RNA tube only. RNA stabilization for up to 3 days at 18-25°C, stabilization for at least 50 months at -20°C or -70°C. The PAXgene Blood RNA Tube contains an additive that stabilizes the in vivo gene transcription profile by reducing in vitro RNA degradation and minimizing gene induction. Transport to the Facility should be done at the same temperature that the tubes have been stored.
If your sample is from cell culture, pellet cells, take out liquid media, and add 700 ul Trizol, vortex at maximum speed for 1 minute, store at -80°C.
C. RNA, miRNA, cDNA
BioMark HD (Fluidigm)
Specific Target Amplification (STA) is recommended which allows for a multiplexed pre-amplification by using a pool of gene expression assays as the source of the primers. By using the same assays in the pre-amplification reaction as the real-time PCR reaction, only targets of interest are amplified. Taqman Gene Expression: 10 ul of each assay (20X) in separate 96 well plates. For 48.48 arrays fill the left half only. EvaGreen Gene Expression: 10 ul of each primer pair in separate 96 well plates. For 48.48 arrays fill the left half only. If you are doing more than 1 array with the same sample or assay set, the volume is different, contact Facility. The Facility does not do primer optimization for Evagreen Assay . Single Cell Submission: Call Facility to schedule delivery no later than 12 noon.
D. Fragmented and Labelled cRNA, ssDNA
Ship samples overnight on plenty of dry ice, and label both exterior and interior of package with your project ID. The print out of the Request ID should be included in the package. Hetty Rodriguez
Molecular Profiling Facility
68 John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104 Phone: 215-573-6316
To retrieve data for your Work Request, go to https://www.bioinformatics.upenn.edu/fs/ You will need to log in with your Penn key and Password. Applied Biosystems Genotyping and Gene Expression raw data will be provided as sds file, processed images and/or data spreadsheets as appropriate. Fluidigm Genotyping and Gene Expression raw data will be provided as bml file, processed images and/or data spreadsheets as appropriate. Affymetrix Transcript Profiling data will be provided as raw scanner images, raw data, normalized data and/or data spreadsheets as appropriate. Quality control reports are also provided. .dat files are raw scanner image .cel files are raw intensity for each cell, it is often imported into other programs for pre processing, normalization and analysis. .chp files are generated by analyzing the cel files with different algorithms. The algorithm used will preface the chp extension as in mas.chp for the mas 5.0 algorithm or .rma.chp for RMA. .arr AGCC sample file are those with information about fluidics, attributes, barcode, type and library files.