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High-Throughput Nanovolume qPCR

Next-Generation Platform Combines Microarray Throughput with Sensitivity

• David Gelfand
• ,
• Kumar Kastury

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Results

Figure 1. Human reference gene GAPDH assay was dispensed onto the entire chip covering 5,184 wells using the custom nanovolume dispenser, sealed with an adhesive film and cycled for 40 cycles followed by melting curve analysis. Click Image To Enlarge +

Figure 1. Human reference gene GAPDH assay was dispensed onto the entire chip covering 5,184 wells using the custom nanovolume dispenser, sealed with an adhesive film and cycled for 40 cycles followed by melting curve analysis.

Researchers have been able to demonstrate complete reproducibility across 5,184 wells with the SmartChip system. In one test, a reaction mix containing all the components for a GAPDH real-time PCR assay was assembled and dispensed into 5,184 wells in a SmartChip using the system’s custom nanodispenser. Data collected following 40 cycles demonstrated the platform’s ability to simultaneously achieve consistent results across thousands of wells (Figure 1).

Additional tests have demonstrated the SmartChip platform’s sensitivity and dynamic range. In one experiment (Figure 2), researchers showed that the SmartChip system possesses a sensitivity of three copies and a dynamic range of five logs.

Additional testing has confirmed the system’s ability to perform and meet analytical performance specifications, while demonstrating the miniaturization of reaction volumes from 10 µL to 100 nL. Real-time results were generated in under three hours for ~1,000 genes in triplicate with 300–500 ng of starting total RNA. Furthermore, researchers successfully tested total RNA samples derived from matched normal and tumor tissues samples from the lung.

Figure 2. Primer-loaded 1000-gene SmartChip was filled with cDNA sample and master mix or master mix without cDNA as negative control, sealed and cycled for 40 cycles followed by melting curve analysis. Click Image To Enlarge +

Figure 2. Primer-loaded 1000-gene SmartChip was filled with cDNA sample and master mix or master mix without cDNA as negative control, sealed and cycled for 40 cycles followed by melting curve analysis.