Fluidigm real time pcr analysis software 3

Total RNA was isolated from the Longissimus dorsi muscle of 114 samples with RiboPure kit (Ambion; Austin, TX, USA). Total RNA was quantified in a NanoDrop ND-1000 spectrophotometer (NanoDrop Products; Wilmington, DE, USA). The RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription (Applied Biosystems). The cDNA samples were loaded into a Dynamic Array 48.48 chip in a BioMark system (Fluidigm; San Francisco, CA, USA) through a NanoFlex controller following a protocol previously described18 (link). For this experiment, the expressed levels of 48 genes were analysed, and a total of 45 target genes were normalized using the two most stable housekeeping genes (ACTB and TBP). Primers used for the analyses are detailed in Supplementary Table S8. Data were collected using the Fluidigm Real-Time PCR analysis software 3.0.2 (Fluidigm) and analysed using the DAG expression software 1.0.4.1157 (link), applying the relative standard curve method (See Applied Biosystems user bulletin #2). Analyses were performed using the normalized gene-expression levels of each sample and assay. The animals showing abnormal gene expression levels (outliers) were removed and data obtained were normalized if necessary by performing log₂ transformation of the Normalized Quantity (NQ) value. The sex effect was also tested by using a linear model with R programme58 .

Total RNA from microglia collected at 4 h was isolated using the Tri Reagent protocol (Invitrogen) and synthesis of cDNA was carried out using a high-capacity RT kit (Applied Biosystems) as previously described for real-time RT-PCR. Fluidigm reactions were performed by the UIUC Functional Genomics Unit of the W. M. Keck Center using a 96 × 96 chip and included two technical replicates for each combination of sample and assay. Data was acquired using the Fluidigm Real-Time PCR Analysis software 3.0.2 (Fluidigm, San Francisco, CA, United States). Data from Fluidigm runs were manually checked for reaction quality before analysis, and C_t values for each gene target (see Table 1) were normalized to C_t values for the housekeeping gene Gapdh.

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).