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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):

Chart of Avereage RNA yield

  • 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.

Target Labeling Assays for Expression Analysis

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.

Applied Biosystems Image

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):

Expected yield

  • 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.

Reaction Scheme Image

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.

Taqman Results

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 Guide

  • 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.

Bead chips in stock:

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

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.