96.96 dynamic array integrated fluidic circuits chip

High throughput microfluidic qPCR was performed using the 96.96 dynamic array integrated fluidic circuits chip (Fluidigm, San Francisco, CA, United States) combining 96 samples with 96 primer sets in 9216 separate simultaneous qPCR reactions.
An assay master mix was prepared and consisted of 3 μL 2X Assay Loading Reagent (Fluigdim, San Francisco, CA, United States) and 3 μL 10 μM forward and reverse primer.
Then a Pre-sample mix was made of 3 μL TaqMan Gene Expression Master mix (Applied Biosystems, Foster City, CA, United States), 0.3 μL 20X DNA Binding Dye Sample Loading Reagent (Fluidigm, San Francisco, CA, United States), 0.3μL 20 X EvaGreen (Biotium; VWR- Bie & Berntsen, Herlev Denmark), 0.9 μL low TE-buffer and 1.5 μL pre-amplified cDNA.
Samples and primers were loaded in the microfluidic chip according to the manufacturer’s instructions, and the BioMark (Fluidigm, San Francisco, CA, United States) was used to perform the microfluidic qPCR cDNA technical triplicates, no template control, -RT samples, amplification curves, melting curves, and dilution curves all served as different quality controls points for each process and were used in the subsequent data preprocessing. Data were handled by the Fluidigm Real-Time PCR Analysis software 3.0.2 (Fluidigm, San Francisco, CA, United States).

Pre-amplification and qPCR were performed as previously described²³ , following the guidelines of the manufacturer (Fluidigm, USA). Briefly, samples were submitted to sequence-specific preamplification in a mix containing 1.25 µL assay mix (final concentration of 0.2× for each TaqMan assay), 2.5 µL TaqMan PreAmp Master Mix (Applied Biosystems) and 1.25 µL cDNA. The reactions were initiated at 95 °C for 10 min followed by denaturing at 95ºC for 15 s, annealing and amplification at 60ºC for 4 min for 14 cycles. The preamplified products were diluted sixfold prior to qPCR.
The 96 × 96 Dynamic Array Integrated Fluidic Circuits chip (Fluidigm) was loaded according to the manufacturer's protocol with each sample solution (2.25 µL diluted-preamplified cDNA, 2.5 µL of TaqMan Universal PCR Master Mix [Applied Biosystems] and 0.25 µL of Sample Loading Reagent [Fluidigm, USA]); and each assay solution (2.5 µL of 20 × TaqMan Gene Expression Assay [Applied Biosystems] and 2.5 µL of 2 × Assay Loading Reagent [Fluidigm, USA]). The qPCR thermal cycling was performed in the Biomark HD System (Fluidigm, USA) using the protocol TaqMan GE 96 × 96 Standard. All the analysis was performed in four biological replicates and Ct values were calculated from the system’s software (Biomark Real-time PCR Analysis, Fluidigm, USA).