For optimal results, the OpenArray™ genotyping system requires 300 copies per reaction. For genomic DNA template, the concentration and amount of DNA to achieve this depends on the size of the genome of the organism being studied.

If we assume 1pg = 978 Mb, the amounts of DNA required for some sample genomes are listed below:

• Human. At 3.5 pg/genome, 300 haploid copies equal 1ng/hole.
• Drosophila. At 0.18 pg/genome, 300 haploid copies equal 54 pg/hole.
• E. coli. At 0.005 pg/genome, 300 haploid copies equal 1.6 pg/hole.
• Wheat. At 13.8 pg/genome, 300 haploid copies equal 4.1 ng/hole.

The DNA sample usage for human genomic DNA in an OpenArray™ plate can be calculated as follows:

1. 5 µL of volume is needed in each well of the OpenArray™ loading plate.
2. 1 ng of DNA is needed per through-hole (or per reaction).
3. Each through-hole in an OpenArray™ holds a 0.033 µL volume.
4. The 5 µL volume needs to have a final DNA concentration of 30.3 ng/µL.
5. Each well is filled with 3 µL master mix and additives; so, 2 µL are available for DNA. Therefore, a 2:5 dilution must be performed.

• 64-fold savings of master mix
• 6 to 20-fold savings of TaqMan™ assay depending on the layout of assays in the OpenArray™ plate
• 2-fold savings of DNA sample

Researchers can use a variety of flat block thermal cyclers, including the Thermo Electron Px2 and MBS systems, and the Bio-Rad slide tower and DNA engine. Researchers can also use BioTrove's NT Cycler. A 50-cycle, two-step protocol is used, beginning with a 10-minute Taq activation step.

Up to three OpenArray™ plates can be imaged at a time in the BioTrove NT Imager or NT Cycler. Imaging takes approximately 10 minutes (about 3.5 minutes per 3072 reactions).

The NT Imager and NT Cycler are optimized for reading FAM, VIC®, SYBR®, and ROX. Alternative dyes (such as LC-Green, TET, HEX, JOE) can also be used. (VIC® is a registered trademark of Applera Corporation. SYBR® is a registered trademark of Molecular Probes.)

Several corrections are made in the analysis of raw images. These include a flat field correction for the LEDs, a general background subtraction for the OpenArray™ plates, and a spectral correction for the different dyes used.

A variety of methods or DNA kits can be used with the OpenArray™ platform. These include phenol/chloroform extraction, boiling; kits from QIAGEN, Gentra, Clontech; as well as many others.

If DNA concentration is low, researchers can use several methods to increase the concentration before running in the OpenArray™ platform. BioTrove customers routinely concentrate samples using SpeedVac and air-drying. Whole genome amplification (specifically, GenomePhi from GE Healthcare) has been used successfully.

Throughput depends on a variety of factors, including the thermal cycler used, instrumentation for adding samples to master mix, and how many individuals are performing the process. For example, a single, full-time employee can set up, load, cycle, and image 32 OpenArray™ plates in a single day using a single OpenArray™ AutoLoader, NT Imager, and Bio-Rad slide tower thermal cycler. This amounts to approximately 98,000 TaqMan® genotyping reactions in a single day.

BioTrove provides proprietary software for genotyping. The OpenArray™ SNP Genotyping application is a project-based software in which data from different imaging runs can be analyzed and managed together. There are multiple methods included for genotype calling, data organization, and display.

BioTrove also has a project-based software for Endpoint Analysis. This software makes plus and minus calls indicating detection of a pathogenic or GMO sequence. Most of the features in the OpenArray™ SNP Genotyping software are also available in the Endpoint Analysis software. In addition, thresholding and display options are available as well as the ability to look at internal control templates using a second dye probe.

All data (both raw and analyzed) can be exported in .xml or .csv format from the OpenArray™ application software for use in third-party software. Genotype calls can also be exported to a genotype table and manipulated in spreadsheet software.

Use of SNPs markers instead of microsatellites is a common practice now. Microsatellites are more informative on average than SNPs; but, they are more prone to mutation and have a more laborious genotyping workflow. The use of a large number of informative SNPs would provide all the information that microsatellites have but with a cheaper, easier, quicker, and more reliable genotyping method.

The OpenArray™ platform offers several advantages over traditional microsatellite genotyping. First and foremost, there is a significant labor savings. Microsatellites typically involve a PCR step, a clean-up step, and a length-based separation step. SNP genotyping on the OpenArray™ is a single-step homogeneous reaction. Additionally, there is a significant reagent savings as well as the time value of information.

OpenArray™ Real-Time has been optimized to use SYBR® Green chemistry. SYBR® Green I is a dye that binds to the minor groove of double-stranded DNA. Used in conjunction with PCR primers, the amount of PCR product can be monitored as it accumulates. The fluorescent intensity will increase proportionally to the amount of product created.

Currently, we use only SYBR®-based assays in our Real-Time system. A Real-Time TaqMan®-based product is not anticipated any time in the near future.

1. Researchers select assays from the BioTrove library or send primer sequences to BioTrove.
2. BioTrove loads the primer pairs in the through-holes of the OpenArray™ plates in a specific layout, then dries them down.
3. BioTrove ships the preformatted OpenArray™ plates containing the dried-down assays to the researcher.
4. The researcher loads OpenArray™ plates with sample and master mix using the OpenArray™ AutoLoader.
5. The OpenArray™ plates are thermal cycled and imaged in the OpenArray™ NT Cycler.
6. OpenArray™ Real-Time software generates amplification curves and melt curves and organizes key data, such as calculated C_t values, in tables.

• LightCycler® FastStart DNA Master SYBR® Green I, available from Roche Applied Science (catalog number 03003230001)
• BSA at a final concentration of 1 mg/µL
• Glycerol at a final concentration of 0.5%
• Pluronic F68 at a final concentration of 0.2%
• MgCl₂ at 3.5 mM
• Deionized formamide at a final concentration of 8%

There is great flexibility in how assays and samples can be arranged on OpenArray™ plates.

In the most common layout, assays are placed in 56 through-holes of each 8 x 8 subarray. (Eight through-holes are intentionally left blank.) Then, researchers load one sample on each of the 48 subarrays with the OpenArray™ AutoLoader. This configuration is for 56 assays against 48 samples on a single plate. In the diagram below, different colored squares represent unique samples. The round, colored dots represent each assay. Black dots represent through-holes that do not have assays.

Another possible configuration is 112 assays and 24 samples per OpenArray™ plate. In this case, researchers load 24 samples to two subarrays. Since each subarray has 56 active through-holes, a total of 112 assays are loaded, one per through-hole.

In the extreme case, 2688 assays can be placed on each OpenArray™ plate. Researchers load a single cDNA sample to all 48 subarrays.

BioTrove is also working to enable an OpenArray™ plate to be loaded with 28 assays, which can be run against 96 samples.

cDNA or DNA templates can be analyzed on the OpenArray™ platform. The cDNA source can be total RNA derived from various sources, including frozen or paraffin-embedded tissues.

The preferred method for cDNA generation is a random hexamer-based reverse transcription. The most robust and reliable kit is the High-Capacity cDNA Reverse Transcription Kit available from Applied Biosystems. However, our Applications Specialists can work with you to determine the best reverse transcription kit for your needs.

For optimal results, the OpenArray™ Real-Time platform requires 100 copies per reaction. For genomic template, the concentration and amount of cDNA or RNA to achieve this depends on the size of the genome of the organism being studied. For human samples, this is approximately 1 ng per through-hole.

The minimum amount of total RNA sample required for reverse transcription using BioTrove's recommended protocol is 10 µL @ 250 ng/µL (2.5 µg total). The amount of cDNA generated from this amount of total RNA is enough to run up to 12 replicates.

OpenArray™ plates accomodate the number of replicates researchers require. BioTrove recommends that at least three replicates are run for each sample.

The number and type of controls needed is dependent upon the experiments being performed. BioTrove recommends that all qPCR assays contain at least one no template control (NTC) to ensure that amplification is not being generated from a contaminant or from primer-dimers.

If other controls are necessary, BioTrove has a panel of endogenous control genes from which researchers may select. For example, for comparative quantification, a number of reference genes may be needed. Standard curves for some types of control templates may be necessary. BioTrove reference genes can be used to normalize amplification of the unknown samples by the C_t method.

BioTrove does not routinely include 18S or GAPDH. BioTrove offers a panel of 13 endogenous control assays that can be included upon request.

Our optimized protocol is a 36-cycle, three-step PCR program. After completion, a melting (or dissociation) curve is generated.

Beginning with a 10-minute Taq activation step, the three steps are 95°, 55°, and 72° C. The melt or dissociation curve is performed over a 30-degree window, from 65° to 95° C. Our Applications Specialists can modify and optimize the cycling protocol if necessary.

Data collection takes place each cycle at 72° C, i.e., during the extension step of the cycling protocol.

Three OpenArray™ plates can be run at simultaneously. One or two plates can also be run; but, in order to provide uniform thermal distribution, all three positions on the thermal block must be occupied.

BioTrove recommends that researchers place MicroSlides thermal blanks on the block where an OpenArray™ plate is not being run. Purchase MicroSlides from VWR, catalog number 48327-000.

Data from about 9000 real-time reactions can be obtained in 3.5 hours when three OpenArray™ plates are run together in the OpenArray™ NT Cycler. A single technician can set up, run, and analyze three instrument runs per day, yielding data for around 27,000 qPCR reactions.

BioTrove provides its own analysis software for Real-Time PCR with the following features:

• Easily imports assay and sample information
• Automatically calculates C_t and T_m values as well as quality scores
• Ouputs files in a database-friendly .csv format
• Provides graphical display of both raw and normalized amplification and melting curves
• Can compute delta C_t and delta delta C_t
• Manages data from different cycling runs according to researcher needs through a project-based structure

Data can be exported for use in other third party analysis software. The software uses files in .xml format. All data can be exported as a .csv file, including raw amplification intensity data, baseline subtracted amplification data, melting curve raw intensity data, and -dF/dT melting curve data. Data from individual OpenArray™ plates can also be moved as discrete units between different projects.

Baseline subtraction is used to normalize the fluorescence amplification curve.

Typically, a passive dye like ROX would be used in a plate-based system to correct for volume differences. However, since OpenArray™ through-holes load through self-metering capillary forces with uniform volume, this was found to be unnecessary.

If the OpenArray™ Real-Time platform does not detect a meaningful increase in fluorescence, a -1.0 default C_t value is assigned. It means the platform was unable to detect any amplification event. If desired, this value can be modified in the software.

Ideally, the researchers design their own primers to the regions of interest for MSP studies. For this purpose, we recommend Methyl Primer Express (free software available from ABI) or http://www.urogene.org/methprimer/index1.html. In both applications, the important steps in designing sequence-specific methylation primers are automated. All that is needed is to import the sequence, then the software will find all relevant CpG islands and provide primer designs based on selected target sequences and melting temperature settings.

Please e-mail support@biotrove.com for further information.

BioTrove recommend two pairs of primers for msPCR, one specific for modified and methylated DNA (Mpair) and the other for modified and unmethylated DNA (Upair). We also recommend designing several primer pairs per region of interest, looking at different CpG islands.