Materials Needed

•	96- or 384-well StellARray™ Kit
•	Optical adhesive plate covers (as an alternative use an optical heat seal plate cover and heat sealer) and optional Compression Pads (for 96-well plates)
•	2 X SYBR® Green Master Mix - 'hot start' recommended
•	Nuclease-Free dH2O
•	cDNA / gDNA samples
•	Laboratory tubes, pipetters, tips, gloves, etc.

Step 1: Calculate Reagent Set-up Volumes.
Please use our Reagent Calculator to calculate the amount of Super Mix (and subsequently the Sample-Specific Working Solutions) to be prepared for your experiment. For example, we recommend a MINIMUM of 3 'Control' samples versus 3 'Experimental' samples for use in our Global Pattern Recognition™ (GPR) software (but, of course you will likely generate more statistically reliable data using more samples per group). To generate a recipe specific to this 3 versus 3 (or 6 sample) experiment you must first select the appropriate Plate Format (384-, 96-, or 96-well Fast) and then input the number of plates (in this example there are a total of 6 plates for a 3 vs. 3 experiment) and then click 'Go!' . You will generate a short list of components for the formulation of a "Super Mix". The Super Mix enables you to distribute one common reagent to each individual, sample-specific, working solution. By minimizing pipetting steps and applying the same Super Mix to each sample you will generate less technical variability between samples.

Step 2: Prepare Sample-Specific Working Solutions.
Below the Super Mix recipe you will see the individual Sample-Specific Working Solution recipe. Following the indicated volumes of each item, you will prepare the solutions to be added to your StellARrays™. The 'Template' component refers to the sample-specific cDNA (or gDNA) to be analyzed. Typically, this solution is a 1:10 or 1:20 dilution of your cDNA reaction in nuclease-free dH2O. For gDNA analysis we recommend 400ng per 96-well plate or 1500ng per 384-well plate. Be aware that the recipes include a predetermined excess thus minimizing the chance of a shortage due to pipetting errors. Note: We recommend the use of 'hot start' SYBR® Green Master Mixes. Use the appropriate Master Mix / Formulation recommended for your specific qPCR instrument.

Step 3: Pipet Sample-Specific Working Solutions into StellARrays™.
Carefully peel off the foil seal from the StellARray™ and then distribute the Sample-Specific Working Solution to each well. Use 10ul per well for both 384-well or 96-well Fast plates or 20ul per well for standard 96-well plates.

Note: We have had success using multi-channel pipetters and solution troughs for more rapid distribution of reagent to 384-well plates. While this requires a careful 'hand' you will become proficient very quickly. Take care to aspirate slowly from the trough to minimize variable volume deliveries. As the volume level in the trough decreases, take care to ensure that all pipet tips are filled prior to delivering reagent to the wells. If you are missing reagent in some tips, replace the aspirated volume back into the trough, tilt the trough to collect the remaining reagent, and finish the delivery using a single-channel pipetter.

Step 4: Seal the Filled StellARrays™.
Apply an optical seal to each StellARray™. Evaporation is your enemy.

Note: We recommend heat sealing the 384-well plates or use optical adhesive seals. We have found that the use of a Compression Pad on a 96-well plate can reduce evaporative volume losses caused by thermal cycling when using an adhesive optical cover.

Step 5: Spin and Soak the Filled StellARrays™.
Quickly centrifuge (or hand-spin) the reagent to the well bottoms. Take care to avoid dislodging the solution onto the optical seal if you hand-spin the plates. Allow the filled plate(s) to 'soak' at room temperature (or 4°C) for at least 15 minutes prior to loading into the qPCR instrument.

Note: Ensure that there are no bubbles trapped at the bottom of the wells. Bubbles on the surface are not a problem as they will disappear once the solutions get heated during the PCR cycling. Bubbles on the bottom may interfere with the dissolving of the stabilized primers as well as the thermal cycling.

Step 6: Generate the qPCR Data.
Follow the recommendations from the Master Mix manufacturer for proper thermal cycling conditions. All StellARrays™ have been designed to allow the use of an annealing temperature of 60°C. Recommended Cycling Parameters:

Standard PCR Conditions

1)	One (1) cycle of 50°C for 2 minutes (helps to dissolve primers)
2)	One (1) cycle of the recommended Master Mix Specific Hot Start Temperature and Time
3)	Forty (40) cycles of: 95°C for 15 seconds 60°C for 1 minute
4)	Dissociation curve (Melt curve) refer to instrument specifications

Fast PCR Conditions

1)	One (1) cycle of 50°C for 1 minute (helps to dissolve primers)
2)	One (1) cycle of the recommended Master Mix Specific Hot Start Temperature and Time
3)	Forty (40) cycles of: 95°C for 1 second 60°C for 20 seconds
4)	Dissociation curve (Melt curve) refer to instrument specifications

Step 7: Analyze the qPCR Data.
Analyze the raw qPCR data as recommended by the instrument manufacturer. For Threshold-based (Ct) instruments set a common threshold value for all StellARrays™ after evaluating the amplification curves. Typically, a value of 0.15 to 0.2 will be satisfactory for use across all StellARrays™ derived from the same Super Mix.

Step 8: Collate the qPCR Data.
Export the data to a spreadsheet (e.g. Excel).

Step 9: GPR the qPCR Data.
Go online to www.bhbio.com and navigate to the GPR Analysis Portal and follow the upload and analysis procedure.

Step 10: Evaluate the Results.
GPR 'hitters' are automatically analyzed to determine possible pathway-associations and a list is provided for evaluation. This may reveal a 'new' biological context and a new StellARray™ kit may be ordered based on this new context enabling objective refinement of future experiments.