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Real-Time PCR Questions
- Assay chemistries?
- Assay design?
- Layout of assays and samples?
- Sample sources?
- Generation of cDNA?
- Sample concentrations and amounts?
- Replicates?
- Controls?
- Workflow?
- Throughput?
- Analysis Software?
The OpenArray® qPCR Platform has been optimized for use with both SYBR® Green I (Custom OpenArray SYG & Pathways) and TaqMan® chemistry (Custom OpenArray DLP).
For our OpenArray SYG plates assays are designed by the customer and the primer sequences are sent to BioTrove. We suggest that you design the primers based on a few simple rules:
- Amplicon size (70-300bp)
- Primer length (18-24nt)
- Melting Temperature (60-65°C)
- Use Primer3 website
For our OpenArray DLP plates TaqMan® assays should be bought by the customer and sent to Biotrove.
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 
for further information.
What is the workflow for using the OpenArray Real-Time platform?
- Researchers select assays. Select from Pathways, Design Custom SYBR® and send sequences or send TaqMan® assays and BioTrove loads the assays in the through-holes of the OpenArray plates in a specific layout, then dries them down.
- BioTrove ships the preformatted OpenArray plates containing the dried-down assays to the researcher. The researcher loads OpenArray plates with sample and master mix using the OpenArray AutoLoader.
- The OpenArray plates are thermal cycled and imaged in the OpenArray NT Cycler. The OpenArray Real-Time software generates amplification curves and melt curves and organizes key data, such as calculated Ct values, in tables.
What is the layout of assays and samples on each OpenArray Real-Time plate?
What template sources can be run in the OpenArray Real-Time Analysis platform?
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.
What method is used to generate cDNA?
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.
What cDNA/RNA concentrations and amounts are needed for the OpenArray Real-Time platform?
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.
How many replicates are performed on the OpenArray Real-Time system?
OpenArray plates accomodate the number of replicates researchers require. BioTrove recommends that at least three replicates are run for each sample.
What controls are needed in a BioTrove qPCR reaction?
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 
Ct method.
At what temperature is the fluorescence intensity measured?
Data collection takes place each cycle at 72° C, i.e., during the extension step of the cycling protocol.
How many OpenArray plates can be run in an OpenArray NT cycler at one time?
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.
What is the throughput of the OpenArray Real-Time platform?
Running OpenArray SYG plates will allow you to perform 24,000+ reactions per day with a little over 1 hr of hands on time. If you run OpenArray DLP plates you will be able to capture 32,000+ data points per day with about 1.5 hrs of hands on time.
What software is used to analyze the gene expression data?
BioTrove provides its own analysis software for Real-Time PCR with the following features:
- Easily imports assay and sample information
- Automatically calculates Ct and Tm 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 Ct and delta delta Ct
- Manages data from different cycling runs according to researcher needs through a project-based structure
Can I access the raw data? What file formats are available?
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.
What methods does BioTrove use to normalize data?
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.
What is a -1.0 Ct value in the software?
If the OpenArray Real-Time platform does not detect a meaningful increase in fluorescence, a -1.0 default Ct value is assigned. It means the platform was unable to detect any amplification event. If desired, this value can be modified in the software.
How many different types of probe sets are recommended for each promoter for qMSP?
BioTrove recommend two pairs of primers for qMSP, 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.