Access to Technologies & Collaborations Behavior Core Cell Based Screening Core Flow Cytometry Genomics Core High Throughput Screening (HTS) High Performance Computing Molecular Screening Center & NIH Molecular Libraries Production Centers Network Nuclear Magnetic Resonance Proteomics & Mass Spectrometry Scripps Energy & Materials Center Macromolecular X-ray Crystallography Licensing & Technology Transfer

Genomics

The Scripps Florida Genomics Core was established to enable access by Scripps Florida investigators to the latest technologies for Next Generation Sequencing, gene expression analysis and genotyping

The Genomics Core maintains and operates four primary technology platforms: Genomics Technology

Affymetrix GeneChip
Illumina Genome Analyzer IIx
Illumina Beadstation
ABI 7900 HT Real-Time PCR

These technologies allow the core to support a wide array of experimental approaches including:

Whole gene expression profiling (over 30 organisms available)
Splice variant quantification
Copy number variation
Secondary validation of array data
Whole genome association studies
ChIP-seq analysis

These technologies allow the core to support a wide array of experimental approaches including:

Whole gene expression profiling (over 30 organisms available)
Splice variant quantification
Copy number variation
Secondary validation of array data
Whole genome association studies
ChIP-seq analysis

For information contact:	Brandon Young
Telephone:	561-228-2751
Email:	byoung@scripps.edu

Gene Expression Analysis Technologies

Gene Expression Analysis

Affymetrix GeneChips Whole genome expression chips are 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.

Applied Biosystems Real Time PCR

Real Time PCR (RT-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.

Illumina BeadChips

Beadchips are designed to look at expression of either entire genomes or focused gene sets on a custom panel, allowing for higher confidence target discovery, disease classification, and pathway studies. Platform features high redundancy probesets and low sample input.

Whole Gene Expression

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. The data can be used to interrogate differences stemming from drug treatment, RNAi, gene knockouts and natural biological effects.

Each sample is processed on a single chip on the Affymetrix GeneChip and in either 6, 8, or 12 samples on the Illumina BeadArray.

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):

It is recommended to use Trizol (Invitrogen part #15596018)extraction and cleanup with Qiagen RNeasy kit (Part # 74106, DNAse optional).
The core has found consistant success using this standard protocol.
QA/QC RNA using the Nanodrop for quantification and the Bioanalyzer or agarose gel for qualitative assessment.
The required amount of RNA is 2μg. If you do not have this amount, then contact the Genomics Core for alternative options.

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:

Affymetrix Mouse 430 plus 2.0, or Mouse Gene ST 1.0
Affymetrix Human U133, or Human Gene ST 1.0
All other chips will require additional time for ordering.

Reaction Scheme

The second strand copy is then synthesized to make double stranded DNA.
In Vitro Transcription of the DNA is performed to produce labeled cRNA.
This cRNA is then fragmented to an average size of 100 bp.
The labeled and fragmented cRNA is then added to the chip and hybridized overnight.
The chip is then washed, stained and scanned.
Preliminary data analysis consisting of QC metrics will be run by the Genomics Core.

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 located in the Genomics Core Building B room B111. 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):

It is recommended to use Trizol (Invitrogen part #15596018)extraction and cleanup with Qiagen RNeasy kit (Part # 74106, DNAse optional).
The core has found consistant success using this standard protocol.

Reaction Scheme

RNA is reverse transcribed into cDNA.
cDNA is added to reaction with gene specific primers.
Detection molecule in master mix is either SYBER green (non specific) or Taqman probe (target specific).
Reactions are amplified on ABI 7900HT.
Reaction is monitored after every extension step for fluorescence.
Fluorescence signal is plotted versus cycle number.
Detection of cycle number is related to absolute quantity of mRNA.
ABI software performs all calculations.

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):

It is recommended to use Trizol (Invitrogen part #15596018)extraction and cleanup with Qiagen RNeasy kit (Part # 74106, DNAse optional).
The core has found consistant success using this standard protocol.
QA/QC RNA using the Nanodrop for quantification and the Bioanalyzer or agarose gel for qualitative assessment.
The required amount of RNA is 2μg. If you do not have this amount, then contact the Genomics Core for alternative options.

RNA Submission

Bead chips in stock:

Mouse Whole Genome Array
Human Whole Genome Array
All other chips will require additional time for ordering.

Reaction Scheme

A cDNA copy is made of the RNA template using poly dT primers.
The second strand copy is then synthesized to make double stranded DNA.
In Vitro Transcription of the DNA is performed to produce labeled cRNA.
This cRNA is then fragmented to an average size of 100 bp.
The labeled and fragmented cRNA is then added to the chip and hybridized overnight.
The chip is then washed, stained and scanned.
Preliminary data analysis consisting of QC metrics will be run by the Genomics Core.

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 Building B room B111. 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

**********************************

Genotyping Technologies (SECONDARY LINK ON PAGE)

Next Generation Sequencing

Affymetrix GeneChips

Applied Biosystems Real Time PCR

Illumina BeadChips

Affymetrix Mapping Array Protocol

Mapping arrays are designed to provide genotyping information on the organism of choice. This data can be used for linkage analysis, haplotype mapping, association, and copy number variation. Affymetrix offers multiple levels of SNP densities to suit the needs of each investigator.

To view products, SNPs and mapping densities of available Affymetrix arrays, click here.

DNA Extraction

DNA extraction is performed by the investigators lab.The Required amount of DNA is 1μg. If you do not have this amount, then contact the Genomics Core for alternative options.

DNA Submission

To provide accurate results the initial DNA 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.

Mapping chips in stock

Mouse 5K
Human 500K
Human 1M
All other chips will require additional time for ordering.

Reaction Scheme

In separate reactions DNA is restriction digested with Nsp I and Sty I.
Complementary adaptors for each enzyme are added to digested DNA and ligated.
Multiple PCR reactions are performed to amplify the ligated DNA fragments.
PCR products are pooled and purified.
Purified products are then fragmented and end labeled with Biotin.
Biotinylated DNA fragments are then added to the chip and hybridized overnight.
The chip is then washed, stained and scanned.
Preliminary data analysis consisting of QC metrics will be run by the Genomics Core.

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

Experiment Cost

Applied Biosystems PCR Protocol

Genetic mapping is designed to provide genotyping information on the organism of choice. This data can be used for linkage analysis, haplotype mapping, association, and copy number variation. Applied Biosystems offers single SNP density. PCR is a very useful as a means to validate SNPs identified by Affymetrix or Illumina mapping arrays. All assays are provided in single tube master mix format include labeled primers and Taq.

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 the validated SNP genotyping assays availble from ABI refer to their website.

Reaction Scheme

DNA is added to reaction with SNP specific reporter labeled primers.
Multiple detection molecules are master mix Allele A -Vic, Allele B - FAM.
PCR Reactions are amplified in a standard thermocycler.
Reaction is read as an end point reaction upon completion of PCR steps.
Fluorescence signal is detected by reading on ABI 7900HT.
Detection VIC and FAM determines genotype AA, AB, or BB.
ABI software performs all calculations.

Viewing Your Data

Analysis of Your Data

Experiment Cost

Illumina Mapping Array Protocol

Mapping arrays are designed to provide genotyping information on the organism of choice. This data can be used for linkage analysis, haplotype mapping, association, and copy number variation. The Genomics Core offers multiple levels of SNP densities to suit the needs of each investigator.

Illumina genotyping chips come in multiple densities. The high density chip (500K-1M SNPs) is run as one reaction, with the medium density (500-2000 SNPs) run in a 96-well format.

To view products, SNPs and mapping densities of available Affymetrix arrays, click here.

DNA Extraction

DNA extraction and purification is performed by the investigators lab. The required amount of DNA is 250ng. If you do not have this amount, then contact the Genomics Core for alternative options.

DNA Submission

Mapping chips in stock

Mouse Medium and Low Density Arrays
Human 500K SNP Array
Human 1M SNP Array
All other chips will require additional time for ordering.

Reaction Scheme

DNA is activated for binding to paramagnetic particles.
Activated DNA is combined with oligos and paramagnetic beads.
Extend, ligate and cleanup SNP specific oligos.
Amplify the SNP specific oligos with universal dye-labeled PCR primers.
Purify labeled DNA and hybridize to array overnight.
Wash and stain and scan SAM or beadchip.
Preliminary data analysis consisting of QC metrics will be run by the Genomics Core.

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

Experiment Cost

**********************************

Frequently Asked Questions (SECONDARY LINK ON PAGE)

Does the core provide RNA or DNA extraction services?
What are the sample quality guidelines?
Does the Genomics Core run quality assurance checks on submitted samples?
How are samples submitted?
How long until I get my results?
How long do you store experimental data?
What are the costs for each experiment?
Do you accept projects from outside institutions?
What are the costs for each experiment?
Do you accept projects from outside institutions?

Does the core provide RNA or DNA extraction services? Individual investigators are responsible for extracting nucleic acid.
What are the sample quality guidelines?  In order to ensure the most accurate results all samples should be checked for concentration, A260/280 as well as gel electrophoresis prior to submission.
Does the Genomics Core run quality assurance checks on submitted samples?  The Core will check all submitted samples. If any samples fail QC at this point the investigator will be notified. The investigator will then have the option to continue with the experiment or submit new samples. The core cannot make any guarantees for the quality of data that is generated using samples that do not pass initial QC check points.
How are samples submitted?  Samples are submitted directly to the Genomics Core in FR-1 room 113M bench 20. For a complete list of submission procedures click here .
How long until I get my results?  Typical turnaround time is 2 weeks from time of receipt of samples and sample submission form.
Do you perform data analysis?  The core will perform preliminary data analysis. If further analysis is required you may use the shared data analysis computer which includes multiple software tools. In addition, there are multible, free data analysis tools available at external websites .
How long do you store submitted samples?  Samples will be kept until the experiment is completed at which time the remainder of the sample will be returned or disposed of.
How long do you store experimental data?  Data is backed up on a dedicated storage device and archived indefinitely.
What are the costs for each experiment?  A general price guideline for estimated price can be found here. For final pricing or quotes for large scale projects contact Brandon Young.
Do you accept projects from outside institutions?  Yes the Genomics Core will provide services for institutions other than Scripps Florida. To discuss pricing and experimental timelines contact Brandon Young.

**********************************

External Pricing (SECONDARY LINK ON PAGE)

If you have any questions about core pricing or to get a quote, please contact Brandon Young at: byoung@scripps.edu

**********************************

Submitting Samples for Analysis (SECONDARY LINK ON PAGE)

RNA Submission

Samples will not be processed until a sample submission form has been received, and an experiment release form has been signed by the submitting Investigator. To submit samples, contact Brandon Young. You will be given a login ID and password for our LIMS site via e-mail. This site will allow you to provide information on each sample, choose an assay type, track progress and download your data upon completion of your project.

Samples submitted to the facility should be in the form of total RNA. The Genomics Core highly recommends following the RNA Isolation SOP RNA01. Total RNA samples can be brought to the Building B room B111. Please do not leave your samples unattended without speaking to facility staff member. We recommend calling 228-2751 before bringing your samples to room B111 to make sure someone is available to receive them. All RNAs will be checked for quality before processing for expression.

Requirements for submitted total RNAs are:

Affymetrix

RNA concentration must be greater than or equal to 200ng/μl.
Minimum of 2μg of total RNA is required for each GeneChip.
Tubes must be clearly labeled with the same name indicated on the submission form, as well as the RNA concentration.

Illumina

RNA concentration must be greater than or equal to 100ng/μl.
Minimum of 0.5μg of total RNA is required for each experiment.
Tubes must be clearly labeled with the same name indicated on the submission form, as well as the RNA concentration.   The Investigator will be notified if RNA samples have low concentrations. Low RNA samples such from sources such as single cell, LCM, or small tissues can be processed using a two-cycle amplification. To request a two-cycle amplification experiment or alternative vendor kit, please arrange a meeting with the facility advisor. To set up a meeting contact Brandon Young. Two cycle amplification samples may incur additional cost and may take longer than two weeks to complete. Estimates on cost and timeline will be made after meeting with the facility advisor.

DNA Submission

Samples will not be processed until a sample submission form has been received, and an experiment release form has been signed by the submitting Investigator. To submit samples, contact Brandon Young. RNA samples can be brought to Building B room B111. Please do not leave your samples unattended without speaking to facility staff member. We recommend calling 561-228-2751 before bringing your samples to B111 to make sure someone is available to receive them. All RNAs will be checked for quality on an agarose gel before processing. The investigator will be notified if DNA samples have low concentrations, or do not pass QC.

Requirements for submitted total DNAs are:

Affymetrix

DNA concentration must be greater than or equal to 100ng/μl.
Minimum of 1μg of total RNA is required for each GeneChip.
Tubes must be clearly labeled with the same name indicated on the submission form, as well as the RNA concentration.

Illumina

DNA concentration must be greater than or equal to 100ng/μl.
Minimum of 1 μg of total DNA is required for each experiment.
Tubes must be clearly labeled with the same name indicated on the submission form, as well as the RNA concentration.

Gene Expression Analysis

Genotyping Technologies

Frequently Asked Questions (FAQ)

Submitting Samples for Analysis

Contact & Pricing Information