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The StellARray™ Technology
The Bar Harbor BioTechnology, Inc. StellARray™ technology is centered on the use of quantitative polymerase chain reaction, also known as Real-Time PCR. This technology reveals views into the cell providing scientists with the ability to measure gene expression or genomic DNA copy-number changes. By revealing a 'constellation' of molecular changes, our StellARray™ technology provides a means to define the biological state of the cell such as treated vs. untreated, disease vs. healthy, or mutant vs. non-mutant. Our cost effective system provides you with an efficient way to more fully utilize your Real-Time PCR instrumentation and plate 'real-estate' combined with integrated analytical tools. There's no need to buy a new instrument - use what you have and get more out of it. Each StellARray™ consists of a 96- or 384-well PCR plate with each well containing gene-specific PCR primer pairs. Derived from our proprietary database of over 4.8 million primers, each primer pair undergoes extensive in silico and in chemico validations ensuring specificity and high sensitivity. Through the use of SYBR® Green I detection systems, Bar Harbor Biotechnology, Inc. primer performance standards are achieved or exceeded for each acceptable primer pair. By using a standardized nucleic acid template in concert with strategically designed primers, Bar Harbor Biotechnology, Inc. has developed a process to ensure the inclusion of the highest quality primer pairs.

The Multi-Functional StellARray™
The StellARray™ Technology has been designed to provide the user with multiple functions. Each primer pair has been designed to detect both RNA (as cDNA) and genomic DNA in separate assays. This design approach, while contrary to traditional approaches, provides the user with the direct capability to analyze both gene expression and genomic DNA copy-number variations (CNV) derived from the same primer pairs. Additionally, this provides a means for defining both positive and negative controls while maximizing the use of the plate 'real-estate'. For example, while Bar Harbor Biotechnology, Inc. provides recommendations for DNA-free RNA preparation, the user may choose alternative protocols. All of the primer pairs in a StellARray™ can detect gDNA, therefore any result (using cDNA as template) where a gene is determined to be "undetectable", is a definition of an insignificant gDNA contribution to the overall signal and thus the RNA preparation was gDNA-free. Also, inclusion of a primer pair capable of detecting an intra-genic target serves a dual purpose as both a negative and positive control for the presence of gDNA-specific amplification for both cDNA and gDNA applications. An added feature of this design approach allows the use of gDNA for validations and QC/QA. Circumventing the need to access a source of RNA/cDNA in which all genes are detectable at a predictable level, Bar Harbor Biotechnology, Inc. can reliably determine the performance of each primer pair - evaluating Ct, Dissociation Curve, and gel-based information.

Primer Design and Validation
Derived from our proprietary database of over 4.8 million primers, each primer pair undergoes extensive in silico and in chemico validations ensuring specificity and high sensitivity. Primer design parameters were selected to enhance:

• Organism specificity with minimized SNP interference (where possible)
• Gene specificity
• Amplicon efficiency
• Uniformity of primer Tm's
• A/T-rich 3'-ends of primers
• Elimination of primer-dimer artifacts
• Generation of single-peak dissociation curves
• Detection of single bands of expected size via gel electrophoresis

With the added flexibility of genomic DNA detection capability, Bar Harbor Biotechnology, Inc. can assess primer performance using a standardized nucleic acid template source. The process begins by running quadruplicate reactions of 96 primer pairs in 384-well plates using a hot-start based SYBR® Green I detection system (and a constant amount of gDNA per well) in the Applied Biosystems 7900HT FAST Real-Time PCR System under default conditions followed by acquisition of a standard dissociation curve.

The first round of evaluation begins with the elimination of any primer pair that generates a multi-peak dissociation curve (DC test). Primer sets that pass the DC test are evaluated by the Ct test. Primers pairs are eliminated if the mean Ct of the quadruplicates is greater than the mean Ct of all remaining primer pairs plus three times the standard deviation. The final test is the determination of the amplicon size by agarose gel electrophoresis. Primer pairs are eliminated if the expected amplicon size is not achieved.

Note: Bar Harbor Biotechnology, Inc. primer designs incorporate a feature to ensure that the 3'-end of a primer contains an A/T-rich domain. This decreases the possibility that a mis-priming event can be extended by the polymerase particularly during the early phases of the reaction where primer concentration is high relative to the template. An A/T-rich domain is more sensitive to mismatches and may 'flap free' from the template and thus not serve as a duplexed substrate for the polymerase. Alternatively, for example, if a mis-primed annealing event occurs and the 3'-end is G/C-rich the primer may be stabilized enough to be extended and thus produce a non-specific extension product. By destabilizing the 3'-end the specificity is enhanced but it may adversely affect the amplicon yield. By utilizing stringent criteria for both design and performance successful results are achieved.

Figure 1: Quadruplicate stellar results from a typical primer pair using 3.5ng gDNA as template, SYBR® Green I detection in a 10ul reaction volume, and run on the Applied Biosystems 7900HT Real-Time PCR System under default cycling conditions. A. Amplification curves. B. Corresponding dissociations curves.

StellARray™ Plate Content Configurations
Bar Harbor Biotechnology, Inc. has developed a proprietary system for objectively organizing the gene content of each StellARray™. Each content-defined plate has been configured to query a specific 'biological context' and includes targets (genes) that have an increased likelihood of being informative for that context. In order to provide the most flexible system, Bar Harbor Biotechnology, Inc. continues to develop 96- and 384-well content-defined plates. Additionally, our Core Plate Centric System provides extraordinary flexibility for user-defined plates. Each 96-well core plate is composed of biological context-specific gene sets with many having minimized over-lap between other plates. Various user-defined combinations of those core plates can be chosen to form 'new' 384-well content-defined plates depending on the Real-Time PCR instrument format (96- or 384-well block type), the user can choose:

• Content-defined 96-well StellARrays™
• Content-defined 384-well StellARrays™
• User-defined 384-well 4ByCore StellARrays™ derived from the core 96-well plates
• Custom User-defined 96-well StellARrays™ from our inventory of primer sets
• Custom User-defined 384-well StellARrays™ from our inventory of primer sets
• Custom User-defined 96-well StellARrays™ from non-inventory of primer sets
• Custom User-defined 384-well StellARrays™ from non-inventory of primer sets

How it Works - The Perfect Circle™
Order your StellARrays™ online from Lonza. Once received, use the plates immediately or store at -20°C for future use. Determine the number of samples you plan to run, set aside the plates to be loaded and return any remaining plates to the freezer. Follow the protocol below to collect and analyze the data. If your results reveal a biological context by our GeneSieve™ process, use this information to loop back into the plate selection process, acquire new StellARrays™ that more closely match your results and continue data acquisition. This process comprising the PerfectCircle™ enables you to 'home in' on the true biology.

The StellARray™ Protocol
For convenience, Bar Harbor Biotechnology, Inc. offers a Sample Prep Recommendation Resource and a Reagent Calculator / Protocol with each purchase to aid in the easy set-up of StellARrays™. Simply input your StellARray™ ID code (see your plate barcode ID), the number of plates (samples) to be set up, and click 'Go' - a recipe will drop down along with a simple protocol. While we have tested several commercially available SYBR® Green-based 2X Master mixes (including our own 'homebrew'), for best results use the 2X SYBR® Green I based Master Mix with which you are comfortable and works in your Real-Time PCR instrument. 1. Load and seal the StellARray™. 2. Collect the Real-Time data and configure your data upload file. 3. Upload the data to the Bar Harbor Biotechnology, Inc. GPR Portal for GPR analysis and obtain results.

The StellARray™ Compatibility
Bar Harbor Biotechnology, Inc. provides StellARrays™ manufactured in three types of plates that are compatible with most PCR thermal cycling blocks:

• Eppendorf twin.tec PCR plate 96, semi-skirted
• Eppendorf twin.tec PCR Plate 384, clear
• MIDSCI Fast Cycler PCR Plates, EU 96 X 0.2ml

Please determine the compatibility of the StellARray™ plate type with your particular Real-Time instrument prior to ordering.