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RNA Isolation RNA can be isolated from tissue or cells. RNA extraction and purification is performed by the investigators lab. The chart below provides expected yields (from Qiagen):
RNA Submission To provide accurate results the initial RNA needs to meet basic quality control standards for concentration and purity. Samples not meeting these criteria cannot be guaranteed to produce viable data. Investigators will be notified if any problems prior to beginning any experiments. Samples must be of high quality 260/280 (ratio of 1.7-2.1). Integrity should be examined by gel electrophoresis or Bioanalyzer. Gene chips in stock:
Reaction Scheme
Viewing Your Data Analyzed data will be provided in .txt, .csv, or .xls format available for download directly from our LIMS site. The raw data files will be available along with the analyzed data files. Analysis of your Data A basic, preliminary data analysis will be performed by the Genomics Core staff. A dedicated data analysis computer is is located in the Genomics Core room 113M bench 19. Data analysis programs are available on this computer. To reserve this computer, sign up must be done through the Scripps Florida calendar system. If you require more in depth analysis of your data please contact Brandon Young Experiment Cost See Pricing List for general guidelines. To discuss actual prices for particular experiments, contact Brandon Young. Experiments will be processed with basic data analysis within 2 weeks of receipt of samples and sample submission form. You will be notified via e-mail if this is not sufficient time to complete the experiment.
Applied Biosystems Real Time PCR Protocol Real Time PCR is designed to provide a profile of gene expression in a given gene comparing multiple samples. RT PCR can be used to interrogate differences stemming from drug treatment, RNAi, gene knockouts for known targets. RT PCR is also very useful as a means to validate genes identified by Affymetrix or Illumina global gene expression. The equipment for this technology is provided by the Genomics Core in room 113C. The investigator is responsible for ordering all reagents and performing all experiments. The Genomics Core will help with experimental design as well as provide technical advice upon request. An online calendar is used to sign up for time on the RT PCR machine. Be sure to use only the time you are signed up for. If you are going to use more time than you signed up for it is your responsibility to contact the next user and make sure it is acceptable to them. If you do not contact them, they have the right to stop your experiment before completion. If continued problems arise from any user the Genomics Core reserves the right to refuse use to that user. To reserve time on the instrument sign up using the Scripps Florida calendar system. Prior to use of this equipment you must be properly trained by the Genomics Core. To set up a training time, e-mail Brandon Young. To view validated Gene Expression assays for use with the ABI Real Time system, click here.
For specific Taqman assays provided by ABI - type your gene of interest into the orange search box on the left hand side of the screen.
RNA Isolation RNA can be isolated from tissue or cells. RNA extraction and purification is performed by the investigators lab.
The chart below provides expected yields (from Qiagen):
Reaction Scheme
Typical results for Taqman RT PCR run. The green line represents the cycle threshold for the beginning of log phase amplification. The relationship between cycle numbers is 2N, where N =the difference in cycle number between two samples. For example sample 1 crossed the threshold line at 10 cycles, sample 2 at 11 cycles. If sample 1 is considered a control then the fold difference between the other samples and sample 1 can be calculated. Sample 1 – Sample 2 = 1 cycle, which is 21 = 2. This means there is a 2 fold lower amount of mRNA present for the gene of interest in sample 2 when compared to sample 1.
Viewing Your Data Data files are saved on a network shared drive in a folder provided for each individual (Gentech_RTPCR_name). This shared drive can be mapped to any computer at Scripps Florida. If you need help mapping a drive contact the IT department at flhelpdesk@scripps.edu. Analysis of your Data Analysis is performed by the user with free ABI SDS software. The software is available on a disc kept in the Genomics core. If you require more in depth analysis of your data please contact Brandon Young. Experiment Cost Pricing is determined based on average usage per year per lab. In order to use the ABI Real Time System, a lab must purchase time on the machine. For specifics on the cost structure, see our Pricing List.
Illumina Gene Expression Protocol Whole genome expression is designed to provide a complete profile of gene expression in a given sample. The levels of activation and inactivation of every gene can be used to compare multiple samples. GeneChips can be used to interrogate differences stemming from drug treatment, RNAi, gene knockouts and natural biological effects. Illumina whole genome expression chips are unique in that 6-8 samples are hybridized on a single chip, using a low starting amount of RNA. To view the genes interrogated by the Illumina microarrays, refer to this link. RNA Isolation RNA can be isolated from tissue or cells. RNA extraction and purification is performed by the investigators lab. The chart below provides expected yields (from Qiagen):
RNA Submission To provide accurate results the initial RNA needs to meet basic quality control standards for concentration and purity. Samples not meeting these criteria cannot be guaranteed to produce viable data. Investigators will be notified if any problems prior to beginning any experiments. Samples must be of high quality 260/280 (ratio of 1.7-2.1). Integrity should be examined by gel electrophoresis or Bioanalyzer. Bead chips in stock:
Reaction Scheme
Viewing Your Data Analyzed data will be provided in .txt, .csv, or .xls format available for download directly from our LIMS site. The raw data files will be available along with the analyzed data files. Analysis of your Data A basic, preliminary data analysis will be performed by the Genomics Core staff. A dedicated data analysis computer is is located in the Genomics Core room 113M bench 19. Data analysis programs are available on this computer. To reserve this computer, sign up must be done through the Scripps Florida calendar system. If you require more in depth analysis of your data please contact Brandon Young. Experiment Cost See Pricing List for general guidelines. To discuss actual prices for particular experiments, contact Brandon Young. Experiments will be processed with basic data analysis within 2 weeks of receipt of samples and sample submission form. You will be notified via e-mail if this is not sufficient time to complete the experiment. |
