A high throughput technology that allows detection of thousands of genes simultaneously for analyzing gene expression. This technology has especially become attractive as it enables the scientist to study many genes in a single experiment quickly and efficiently especially when one wants to analyze a large repertoire of genes with a small number of samples.

Microarrays may be used to assay gene expression within a single sample or to compare gene expression in two different cell types or tissue samples, such as in healthy and diseased tissue. Because a microarray can be used to examine the expression of hundreds or thousands of genes at once, it promises to revolutionize the way scientists examine gene expression. Scientists are using microarray technology to understand fundamental aspects of growth and development as well as to explore the underlying genetic causes of many human diseases. The study of pathways using expression data has become one of the key areas in System Biology. Scientist world-wide have used microarrays extensively in tumor classification, prognosis prediction, drug development, drug response, tracking disease progression etc.

The Microarray Facility at The Centre for Genomic Application enables scientists to perform gene expression studies from a varied choice of platforms depending upon individual research needs. Scientist at The Centre for Genomic Application are using this technology in comparative genomic hybridization for cancer studies, epigenetic studies, alternate splicing, study of miRNA, and gene expression profiling of clinical samples to understand the disease at the molecular level. The facility offers gene expression services on Affymetrix Gene Chip and Illumina Bead Array platforms. Target validation and mRNA quantification is available using the Roche Light Cycler 480 platform. Detailed microarray data analysis services are available for which you are encouraged to contact the scientists. These services encompass the entire spectrum of experiments starting from experiment design to microarray data analysis.
 

Services Available

• RNA Extraction(This service is only available with expression services only). We offer RNA extraction from a variety of biological samples. All samples must be properly labeled with a unique ID

• Blood samples – Fresh blood stored in anticoagulant and kept in ice should be received within 1-2 hrs from the time of drawing blood .

• Tissue samples - Biopsies or tissues must be stored in either liquid nitrogen or dry ice or RNAlater

• Cultured Cells - Must be stored in RNAlater or in liquid nitrogen

• Leucocytes - Must be stored in RNAlater or in liquid nitrogen

• RNA Quality and Quantity check
  Quantifying RNA : RNA quantification is done by U.V. spectrophotometer or nano drop

• RNA Quality Check using the Agilent Bioanalyzer 2100
  We offer “Lab on a Chip” platform from Agilent that checks the quality of RNA samples. RNA 6000 Nano Assay and RNA 6000 Pico Assay are two assays available for doing RNA quality check.(RNA assay specification)

• Gene Expression using the Affymetric Gene Chip platform (Process flow of Affymetrix Gene Chip platform)
  Researchers are using the Affymetrix Gene Chip® system to more rapidly discover RNA patterns which can better classify disease, understand mechanism-of-action, and predict response to therapy in a number of disease areas.

• 3’ expression arrays- Robust, simple representation focusing on the 3' ends



• Exon/gene Arrays-Genome-wide, whole-transcript coverage for a survey of alternative splicing and gene expression.
  
  List of Affymetrix Arrays: (Click Here)

• Gene Expression using the Illumina Bead Array platform (Process flow)
  

  It is a high throughput platform. The Beadarray Technology makes its preprocessing and quality control different from other microarray technologies. Illumina's multi-sample format lets you assay more than 48,000 transcripts and profile six samples simultaneously on a single HumanWG-6 BeadChip .The HumanRef-8 Bead Chip targets approximately 24,500 well-annotated RefSeq transcripts and enables the interrogation of eight samples in parallel.

  List of Illumina products (Click Here)

• Gene Expression using real time PCR (RT-PCR) quantification (Process Flow)
  

  • Roche LightCycler® 480 is used to analyze cDNA for the purpose of determining relative and absolute quantitation of gene expression. We routinely use this platform to validate Illumina and Affymetrix gene expression results and mRNA quantification of specific gene of interest. In addition to quantifying gene expression, potential applications for real-time PCR include mutation detection, allelic discrimination, viral load estimation and detection of single nucleotide polymorphisms (SNPs). It can also be used for Copy number detection is also one of the application of real time PCR

  • Primer design and synthesis-Design custom primers sets using state of the art software and design parameters
  • Relative quantification and Absolute quantification using
  • SYBR Green chemistry
  • TaqMan probe chemistry
  • Molecular beacon
  • Scorpion probe
  • Locked Nucleic Acid Fluorogenic probe
  • Taqman MGB probe

Microarray Data Analysis Services

TCGA offers a range of bioinformatics analysis options to aid researchers in the interpretation of their data.
Basic Analysis is included with Microarray gene expression and consists of following:

• Raw Data Files: Expression Analysis returns the primary, platform-specific data files and text files that can be incorporated into any analysis software.

• QC Results: This report contains the QC results collected for each sample during target preparation and hybridization.

• Data Summaries and Comparisons: This file assimilates expression data, such as signal intensities and detection calls for every sample in a common study, and includes gene annotation information. The Normalized data is provided in this case after background noise correction.

• Normalization

• Data filtering (Background Reduction).

• Graphical representation of data in various formats (Scatter Plot(s), Pair-wise volcano plot(s), Box plot(s))

• Statistical analysis to identify differentially expressed genes

• Annotation of the differentially expressed gene lists with gene identifiers and functional information

• Principal Component Analysis – enables the identification of outlier samples

• Clustering (hierarchical and non-hierarchical) of the gene expression data. This groups together genes with similar profiles and discovers gene relationships
   

Full Analysis Service (FAS):The Full Analysis Service includes all the components of standard and premium service along with the following analysis options.

• Gene ontology analysis: Assessing the content of differentially expressed gene lists in terms of their predominant biological themes.

• Inference of biological networks from gene expression microarray data.

• Gene set enrichment analysis (GSEA) – Evaluates expression microarray data at the level of gene sets (pathways).

• Discovery of pathway network relationships within the data in the context of known pathways.

• Use of text mining algorithms to extract network relationships within PubMed for the data.

Deliverables
Results from the analysis services are provided on a DVD/CD containing raw data, a quality control report, figures, and analysis files, where applicable.
For expedited delivery, results can be sent to you via our secure FTP server

Consultation
Our bioinformatics experts can make the appropriate recommendations for the development of your data analyses throughout your project. This is from the experimental design stage to ensure adequate statistically robust data is generated, through to offering guidance on the most appropriate and latest cutting edge data analyses methods for your experiment data.

Technology

TCGA Microarray Facility is equipped with latest technology to meet the standards of the present scientific community.

Agilent 2100 bioanalyzer
The Agilent 2100 bioanalyzer is the most successful micro fluidics-based platform available commercially, offering solutions for the analysis of DNA, RNA, proteins and cells. It estimates the quality in a matter of 30 minutes, delivering automated, high quality digital data. This technology utilizes a network of channels and wells that are etched onto glass or polymer chips to build mini-labs. Pressure or electrokinetic forces move pico liter volumes in finely controlled manner through the channels. ‘Lab-on-a-Chip’ enables sample handling, mixing, dilution, electrophoresis and chromatographic separation, staining and detection on single integrated systems. The main advantages of ‘Lab-on-a-Chip’ are ease-of-use, speed of analysis, low sample and reagent consumption and high reproducibility due to standardization and automation.

Agilent 2100 bioanalyzer

Affymetric Gene Chip platform
The Affymetrix GeneChip system consists of the probe/gene microarray, hybridization oven, fluidics station, computer workstation and a scanner .
Oligonucleotides, usually 25-mers, are directly synthesized onto a glass wafer by a combination of semiconductor-based photolithography and solid phase chemical synthesis technologies.Each array contains up to 900,000 different oligos and each oligo is present in millions of copies. Each gene is represented on the array by a series of different oligonucleotide probes.  Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide.The perfect match probe has a sequence exactly complimentary to the particular gene and thus measures the expression of the gene.The mismatch probe differs from the perfect match probe by a single base substitution at the center base position, disturbing the binding of the target gene transcript.

Affymetrix GeneChip System

 This helps to determine the background and nonspecific hybridization that contributes to the signal measured for the perfect match oligo. GeneChip Expression Console subtracts the hybridization intensities of the mismatch probes from those of the perfect match probes to determine the absolute or specific intensity value for each probe set. Probes are chosen based on current information from Genbank and other nucleotide repositories. The sequences on the expression arrays are believed to recognize unique regions of the 3’ end of the gene.

Illumina: beadarray technology
Illumina’s Bead Array Technology is based on 3-micron silica beads that self assemble in microwells .Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences in one of Illumina’s assays. Bead Array technology is utilized in Illumina's iScan System for a broad range of RNA analysis applications.

Illumina Platform  

LightCycler ® 480 Real-Time PCR System
The LightCycler ® 480 Real-Time PCR System is a high throughput gene quantification or genotyping real-time PCR platform with exchangable blocks for 384 samples in multiwell plates. It offers

• enhanced throughput

• compatibility with automation equipment

• maximum flexibility regarding hardware and software

The LightCycler ® 480 Real-Time PCR System can complete a 384 well PCR run in 40 minutes.
The LightCycler ® 480 Real-Time PCR System features a compact and versatile bench top instrument that includes interchangeable thermal block cycler units for 384-mulitwell plate formats.

LightCycler ® 480 Real-Time PCR System

FAQS

1. What are the various Microarray services provided by TCGA?
RNA quality check with Agilent 2100 Bioanalyzer
Gene expression using Affymetrix Gene Chip platform
Gene expression using Illumina Bead Array platform
Gene expression using real time PCR (RT-PCR) quantification
Data Analysis for gene expression studies.

2. How much RNA is to be send for expression studies? At what concentration?
It depends on the type of service you have requested for. Real time quantification requires minimum 1.5 μg of purified RNA. Affymetrix platform requires minimum of 6 μg of RNA and Illumina platform requires 500ng.

3. What is the recommended protocol for RNA isolation?
We do RNA isolation by either TRIzol Method or using Qiagen RNeasy Mini Kit depending on the sample type. For detailed information please contact us at microarray@tcgaresearch.org

4. Is it necessary to check RNA in Denaturing agarose gel systems?
It is preferable that you check your RNA on a denaturing gel. Denaturing agarose gel systems include formaldehyde and MOPs buffer to denature the RNA so that molecules will run by size. The 28S and 18S rRNA bands are visualized by ethidium bromide staining

5. Why is it necessary to purify RNA?
Since contaminants like genomic DNA could hinder at a later steps in a microarray experiment it is necessary to purify RNA. In expression experiments the quality of the expression data depends on the purity and amount of RNA. Our facility has completed extensive research with various purification methods and provides services and suggestions to assist you with producing high quality RNA samples.
6. How should RNA be stored?
RNA must be stored in -20°C for short term storage and for long term -80°C.

7. What are the time durations of these services?
We assure you to give best results in minimum possible time although it entirely depends on the project /service requested.

8. How should I send RNA samples?

RNA samples must be sent in microcentrifuge tubes (preferably cryovials) sealed with parafilm in dry ice or in liquid nitrogen.

9. What is the best way to determine the concentration of my RNA sample?

We recommend estimation on Nanodrop else by spectrophotometer.

10. Do I have to fill out the whole sample sheet?
Yes, you need to fill the submission form.

11. How can I obtain TCGA's Microarray order form?
The order form is available on our website http://www.tcgaresearch.org or call and we will e-mail or fax it to you.

12. What will I be charged if a reaction fails?
This does not happen very often and when it does, we make every effort to determine what went wrong. If our positive controls indicate a processing failure, we will repeat the experiment at no charge. If it appears the sample is the problem, then it will be charged. We will then work with you to assure that subsequent reactions proceed more successfully.
13. How much is the cost of Microarray services?
For details contact commercial@tcgaresearch.org or at microarray@tcgaresearch.org

14. From where do I procure my arrays for expression studies?
TCGA will procure the arrays for you. Regarding reagents and all for other platforms it is project dependent.
15. How do I decide which platform to choose for expression studies?
Selection of a microarray platform depends on many factors like study organism, gene of interest, cost and purpose of study

16. What all do I need to send along with my samples?
You just need to send the filled sample submission form along with your RNA samples

17. How will the data be send to me?
The data will be send to you by courier and in case of urgency we can upload in the FTP server.

18. Can I come to TCGA Microarray Facility to observe my experiment being performed?
As per TCGA policy we do not allow any one in our lab however please feel free to clarify your queries by mailing us at microarray@tcgaresearch.org.

19. How to decide which chemistry to be used for mRNA quantification by real time PCR?
SYBR Green chemistry is sensitive, convenient and cost effective but can also bind to non specific sequences leading to false signals and multiplex reaction are not supported in this chemistry. Probe specific chemistries area more specific and allows multiplex reactions with ease although probe synthesis adds to the cost and complexity. To simplify analysis of your multiplexed samples we suggest taking advantage of the broad spectral range by using dyes whose emission spectra lie far apart. This limits the amount of spectral overlap between the probes, so that color compensation is no longer necessary.

20. Who do I contact for questions related to various expression platforms?
For any kind of assistance and query please feel free to contact at commercial@tcgaresearch.org or at microarray@tcgaresearch.org.

21. How and in which format will I receive my data of mRNA quantification?
Report in adobe acrobat pdf format as well as excel sheet will be provided which will contain detailed analysis.

22. What are the requirements for the purity of RNA for expression studies?
RNA should be highly purified and free from DNA or any other kind of contamination. Quality check reports (bioanalyser report, absorbance) should be send along with the sample else the service (absorbance on request with no additional charge) can be availed form TCGA

23. When dealing with RNA, what should I keep in mind?
RNA isolation procedure should include some important steps both before and after the actual RNA purification. RNA can be stored at -80C for up to one year, but freshly isolated samples are always best. Repeated freeze and thawing of RNA can lead to degradation of RNA.

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