Biomark platform

48.48 Dynamic Array used in the present study are nanofluidic chips able to analyze 48 samples with 48 SNP assays on the BioMark platform (Fluidigm). The BioMark system is used to thermal cycle these nanofluidic chips and image the data in real time.

dPCR was performed on a BioMark platform (Fluidigm Corporation, San Francisco, California) with qdPCR37K integrated fluidic chips, following the manufacturer’s instructions with slight modifications. Instead of using 1.8 μL of DNA template, 2.1 μL of diluted pre-amplified DNA was used in each 6-μL PCR reaction. Patient samples underwent dPCR with each of the four TaqMan assays in a singleplex format using the TaqMan Gene Expression Master Mix (Life Technologies, Carlsbad, California) with a protocol of 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The presence of a single red spot with a sigmoidal amplification curve with a cycle threshold (Ct) value ≤23 in each panel was considered as a positive result.
qPCR was performed with 3 μL of diluted pre-amplified DNA as the template and the TaqMan Gene Expression Master Mix in a total volume of 20 μL on a QuantStudio 7 Flex Real Time PCR System (Life Technologies, Carlsbad, California), following the vendor’s instructions. The PCR parameters were the same as those described above. A Ct value <35 combined with a sigmoidal amplification curve was considered as a positive detection result. All qPCR reactions were performed in triplicate and repeated thrice to monitor reproducibility.

Gene expression in single cells was measured by quantitative multiplex PCR following pre‐amplification using the Biomark platform (Fluidigm) according to the manufacturer's instructions. Data were normalized as described (Livak et al., 2013). Briefly, the data adjustment was as follows: Log2 expression data were extracted through the single‐cell analysis package for all PCR plates, including both C1 and manual isolation data. The data were then categorized as deriving from quiescent or senescent cells prior to subsequent analyses.

Whole-liver tissue samples were homogenized and RNA extracted using an animal tissue RNA purification kit (Norgen, ON, Canda). RNA extracts were tested for overall RNA quantity using a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE). Total RNA was converted to cDNA and pre-amplified prior to real-time qPCR analysis using the Biomark platform (Fluidigm, San Francisco, CA). The data were normalized by median-centering within each sample, followed by median-centering within genes. Primers used for PCR analysis are available in S1 Table.

The plants were either straight or inclined at 35° for 30, 60, 120 or 180 min in the isotropic device. Eight independent biological replicates (individual plants) were produced for each condition. XU and XL cDNA samples were used to monitor the expression of the 153 candidate genes by real-time qPCR at high-throughput. The experiments were performed according to the Fluidigm gene expression protocol without modification using the Biomark platform (Fluidigm, USA) and the 96x96 IFC gene expression (integrated fluidic circuit). Relative quantification was performed using the 2^−∆∆Cq method developed by Pfaffl (2001) (link). Expression levels were related to the geometric mean of the 153 expression levels of the candidate genes proposed by the Bestkeeper reference research software (Vandesompele et al. 2002 (link); Pfaffl et al. 2004 (link)), but using a larger subset of genes. No significant Tukey post hoc HSD was found in the change in gene expression (P < 0.05, data not shown) between qPCR and microarray, which validated the microarray results.