Description
The microarray facility was initiated in 2006 as a central facility at IMBB and as a concerted action of all Departments. The IMBB microarray facility aims to contribute to the progress of ongoing science at the IMBB by providing a number of Genomics and bioinformatics approaches. The facility is in close collaboration with the Biomedical Informatics Laboratory (ICS) that are housed at FORTH . Its mission is to design and deliver, among other, reliable and innovative gene expression services to the IMBB life science community and its collaborators.

Personnel
Scientific advisor

Technician

Infrastructures
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System

The system includes:

• 1. A four-color GeneChip Scanner 3000 7G with AutoLoader that is a 16-bit confocal scanner with a solid state 532nm Green Laser. It provides auto-set laser power and high resolution scanning at pixelations from 2.5 ΅ m down to 0.51 ΅ m. It supports newer applications in gene expression and DNA analysis applications, including the ParAllele Molecular Inversion Probe (MIP) technology highly multiplexed assay for Custom Genotyping.
• 2. Two Fluidics Station 450s that performs washing and staining of the probe arrays in an automated manner . Each station contains four modules, with each module holding one probe array. Every module is controlled independently by the operating software using preprogrammed fluidics protocols.
• 3. One Hybridization Oven 645,that can carry out hybridization of up to 64 arrays at a time
• 4. A computer workstation with quad-core Xeon processors loaded with Affymetrix GeneChip Command Console Software (AGCC) that drives the 2 fluidic stations and the scanner
• 5. Pre-amp and post-amp workstations with GeneChip® Targeted Genotyping Analysis Software and also a 2D handheld barcode reader with each workstation for automated data tracking.

All the necessary Affymetrix software needed for gene expression,mapping, resequencing and targeted genotyping applications are loaded in the workstations.

ScanArray 5000 Scanner

The PerkinElmer ScanArray® 5000Microarray Acquisition System is a scanning laser confocal fluorescence microscope that is used to determine the fluorescence intensity of a glass slide microarray

Scanners features: 

• Wavelength : 4 wavelength choices can be used to support 14 fluorescent dyes.
• Laser Excitation Wavelengths: 488 nm, 543 nm, 594 nm and 633nm ; 488 (SYBR green, Alexa 488, Fluorx, Joe, FITC); 543 (CY3, Alexa 543, TAMRA, Alexa 546); 594 ( Rox , Texas Red, Alexa 594); 633 (Cy5, BODIPY 630). 
• Slide Holder Flexibility: Accommodates a wide range of custom and commercial substrates and can be used with wet or dry samples (with or without cover slips) as well as tissue samples. 
• Sample Size Single slide: Length 75.0 mm - 76.2 mm (2.95" - 3.0") Width 24.6 mm - 25.5 mm (0.97" - 1.02") Thickness 0.93 mm - 1.29 mm (0.037" - 0.051")
• Scanning Field : 22 mm x 73 mm.
• Pixel Resolution: User selectable scanning resolutions: 5, 10, 20, 30 and 50 microns. 
• Emission Filter Wavelengths:   508 nm, 522 nm, 530 nm, 570 nm, 578 nm, 614 nm, 660 nm and 670 nm.
• Sensitivity: < 0.1 fluorescent molecules/µm 2 . 
• Image File Formats: 16 bit TIFF, BMP, JPEG or Raw.
• Barcode Reader: to automatically define scanning parameters and for automated data tracking.

Packard SpotArray 24

The SpotArray 24 is a contact microarray printer including an instrument module, a utility module that provides power supply and contains air and water filters, a reservoir for wash water and waste and a humidity module. In instrument module a robotic arm controls a print head, capable of moving precisely in three axes: x and y and z and holds up to 48 spotting pins that deposit printing material onto slides. The arrayer holds up to 20 coated slides plus 4 blotting slides and delivers samples to be printed from up to 4 384or96well printing plates Up to ~30,000 individual spots of average size can be transferred from the plates to a standard 1 x 3 cm slide After each round of printing, the pins are washed with a pressure-jet pin washer and then vacuum-dried, eliminating contaminations and carryover.

Biomek 2000

The Biomek 2000 is a a flexible liquid handling platform that is also capable to perform a variety of applications like DNA purification and PCR reactions. Tools included with the workstation are:

• Two 8-channel multiple pipettors (2-20 µl and 20-200 µl),
• A single-channel pipettor that can sense liquid levels (100-1000 µl), and
• A wash unit capable of delivering and aspirating larger volumes (>200 µl)
• A 96-Filtration System: for automated Vacuum-Based Separations in Microplate Format.
• A Gripper Tool to move labware and devices around the Biomek worksurface
• Two-side modules for increased capacity
• Labware with 24-well, 96-well, or 384-well formats as well as tubes of various shapes and arrangements.
  

Tecan HS4800 Hybridization Station

The Tecan HS4800 is used for automated hybridization of standard format microarray slides. The advantage is the full automation, i.e. the HS4800 is performing all protocol steps in a highly accurate and reproducible manner. It consists of 12 heating blocks with hybridization chambers on top. The HS4800 controls the temperature and the processing times (hybridization, washing). The hybridization solution (100 ul) is applied by a disposable pipette tip. Due to a highly efficient agitation of the sample within the chamber, the hybridization is faster and more even than under a cover slip. The HS4800 also dries the slides.

NanoDrop® ND-1000

The NanoDrop is a full spectrum (220-750 nm) spectrophotometer that is used for quantitation of RNA, DNA, and protein samples with high accuracy and reproducibility using just 1ul of sample.

MJ Research Real Time PCR Machine

DNA Engine Opticon is a continuous fluorescence detection system used in real-time quantitative PCR (qPCR) applications. This system has an excitation range of 450-495 nm and a detection range of 515-545 nm. It has a heated lid which eliminates the need for oil on top of samples and samples can be run in either 96-well plates or strip tubes. It has a temperature range of 0 to 150°C with an accuracy of ±0.3°C and a ramping speed: up to 3°C per second. A temperature gradient feature is available that allows the user to easily optimize the annealing temperature of the PCR reaction by running each column of wells at a separate temperature. A gradient calculator is built into the software which makes it easy to calculate the well temperature across all 12 columns. The Opticon is compatible with many different chemistries including SYBR® Green I, Molecular Beacons, TaqMan® Probes, Scorpion® Probes, and the Amplifluor® System. Opticon Monitor software facilitates experimental setup, run initiation, run status, and data analysis. software generates graphs and plots data and allows adjustment of threshold values. Graphs can be made using fluorescence versus cycle number, log quantity versus threshold cycle, or both. Besides this unit, additional real-time PCR units are available at various labs within the IMBB (please contact the facility for further info on this).

Methods
Expression Arrays

3' IVT arrays are used for genome-wide gene expression profiling having probe sets targeted to the 3' end of the transcript. Each gene is represented by a series of different 25-mer oligonucleotide probes that are synthesized in pairs, known as perfect match (PM) and mismatch (MM) probes.
Exon arrays have probes distributed across the full length of the gene, and not only the 3' end ,providing a more complete and accurate picture of overall gene expression. With approximately four probes per exon and roughly 40 probes per gene, Exon 1.0 ST Arrays can be used for exon-level and gene-level type of expression analysis.
In Gene 1.0 ST Array each transcript is represented by approximately 26 probes spread across the full length of the gene. Transcript level, but not exon level, expression values can be generated from the data.
A wide range of arrays for over 30 eukaryotic and prokaryotic organisms are available for analysis with 3'based expression arrays. For a complete list of all available 3' expression arrays, click here.

Sample flow for gene expression
Samples RNA ISOLATION TOTAL RNA QUALITY CHECK LABELLING LABELLING QUALITY CHECK HYBRIDIZATION INCUBATION WASHING SCANNING QUALITY CHECK RAW DATA QUALITY CHECK

Samples
Samples are stored in -80C. In particular projects, the user may consider using RNA later an aqueous, non-toxic tissue storage reagent that rapidly permeates tissues to stabilize and protect cellular RNA.

RNA ISOLATION
We routinely use the Qiagen RNeasy kit or Trizol reagent (Invitrogen) for isolating total RNA from cell cultures or tissues. For Affymetrix arrays, we strongly recommend the use of the Qiagen RNeasy kit as the use of the Trizol protocol gives often less optimal results (a recurring problem with Trizol can be the residual ethanol in the eluates ) . If the user wishes to obtain gene expression as well as miRNA expression profiles from the same samples, we recommend the use of TRizol as small RNA fragments (<200nt) will be eliminated with the Qiagen columns.

TOTAL RNA QUALITY CHECK
Isolation of intact RNA is essential for gene expression analysis. Following total RNA isolation, samples will be analyzed for concentration and overall quality ( Check concentration, A260/A280, A260/A230 ) through the use of the NanoDrop ND-1000 Spectrophotometer . The A260/A280 ratio should be maintained close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable). The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band (Figure 1, lane 3). This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Alternatively one could use the Agilent 2100 Bioanalyzer (for integrity) where s amples are detected by their fluorescence and translated into electropherograms or into gel-like images.

LABELLING
3' IVT Express Kit User Manual for 3' IVT arrays
Whole Transcript (WT) Sense Target Labeling Assay for Exon/Gene ST arrays
Whole Transcript (WT) Double-Stranded Target Assay for tiling arrays

LABELLING QUALITY CHECK
The concentration of aRNA solution is determined by measuring 2 ΅ L of the aRNA using a NanoDrop Spectrophotometer . The size distribution of aRNA can be evaluated using an Agilent 2100 bioanalyzer ,or by conventional denaturing agarose gel analysis. The expected aRNA profile in 3' IVT arrays is a distribution of sizes from 250–5500 nt with most of the aRNA between 600–1200 nt while f ragmented aRNA has a distribution of 35–200 nt with a peak at approximately 100-120 nt.
In Gene/Exon arrays ,cDNA is quantified by a NanoDrop Spectrophotometer and should be >5.5 ΅ g, while fragmented ss cDNA that is used for hybridization ,has a peak range of 35–200 nt when analyzed by bioanalyzer.
In tiling arrays ,dsDNA is quantified by a NanoDrop Spectrophotometer and should be >7.5 ΅ g, while fragmented ds cDNA that is used for hybridization , is between 25 to 200 bases, with the peak of the distribution between 25 to 100 bases.

HYBRIDIZATION
Fragmented and labelled aRNA or cDNA samples, depending on array type, are hybridized together with control oligonucleotides on arrays, in amounts specific for the probe array format used.

INCUBATION
Arrays are hybridized for 16 hours into the hybridization oven, set to 45 °C and rotating at 60 rpm.

WASHING
Chips are transferred to a fluidics instrument that performs an automated washing and staining protocol, dedicated to the specific array type . The Fluidics Station 450 can independently process an array using a different fluidics protocol in each of four different modules.

SCANNING
Arrays are scanned using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. The software defines the probe cells and computes intensity for each cell creating a probe array image file with .dat extension.

QUALITY CHECK
Visual inspection of the scanned image in AGCC Viewer, looking for any defects, areas of high background, or areas of low signal. As long as these areas do not represent more than 10% of the total probes for the chip, then the area can be masked and the data points thrown out as outliers without affecting the total quality of the data.
The border around the array, the corner region, the control regions in the center, spiked-in Oligo B2 control are also checked to ensure the hybridization was successful.

RAW DATA
AGCC software aligns a grid on the image to identify the probe cells , and automatically computes probe cell intensities and saves the data to the cell intensity file (CEL).
Expression Console software is used to generate probe set summarization (CHP) files from feature intensity (CEL) files for 3' Expression Arrays, Gene Arrays, and Exon Arrays using MAS5,RMA, or PLIER algorithm and also provides several quality controls metrics.

QUALITY CHECK
Track the progress of the data through grid alignment and cell intensity generation in AGCC viewer. Check the grid alignment and realign the grid, if necessary.
Examine Cell Summary Report of AGCC, for monitoring the performance of the hybridization and grid alignment of arrays. Examine quality controls metrics of Expression Console software for overall chip quality :