Pcr array data analysis web portal

Real-time qPCR analysis for the PCR arrays was performed using the RT² SYBR green qPCR Master Mix (SABiosciences) on a StepOnePlus™ Real-Time PCR System (Life Technologies) as described previously [21 (link)]. cDNA from groups of male mice (n = 4 per group) was tested using the Cytokines & Chemokines RT² Profiler PCR Array (Cat#: PAMM-150ZE), which contains 84 genes related to inflammatory pathways and the immune response (S1 Table). A set of 5 housekeeping genes (HKG) was used as internal controls for standardization between samples: Gusb, Hprt, Hsp90ab1, Gapdh and Actb. Results were analyzed using the PCR Array Data Analysis Web Portal (SABiosciences), followed by Benjami-Hochberg FDR correction.

RD cells in 24-well plates were infected with E7 WT or mutant viruses at an MOI of 10, or a consistent viral genome copy number equating to an MOI of 10 in the WT. Sendai virus and Poly I:C, strong inducers of IFNβ, were separately inoculated as positive controls. Viral RNA titres were confirmed in virus stock preps using qRT-PCR. Cells were harvested 8 h post infection, and RNA was isolated. RNA from six biological replicates was combined, and RNA integrity number was confirmed to be greater than 9.7 for all samples using a 2100 Bioanalyzer (Agilent). cDNA was made using the RT² First Strand cDNA Synthesis kit (Qiagen) with 800 ng RNA per sample. The relative expression of 84 candidate genes was then analysed with pre-made ‘Human Innate & Adaptive Immune Responses PCR Array’ RT² Profiler PCR Arrays (SABiosciences, catalogue number PAHS-052), and RT² SYBR Green Rox Fast Master Mix (Qiagen). Cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Data were normalized and fold changes calculated using the PCR Array Data Analysis Web Portal (SABiosciences).

The spleens of treated mice were harvested on day 6, snap frozen and RNA purified. RNA was converted to cDNA using the RT² first strand kit (Qiagen) and qPCR performed using RT² SYBR Green qPCR mastermix (Qiagen) and RT² profiler PCR array for mouse cytokines and chemokines (Qiagen) as per manufacturer’s protocol. Data analysis was performed using the ΔΔC_T method and SABiosciences PCR Array Data Analysis Web portal.

Clinical data was analysed using GraphPad Prism 5 software. Comparison of means was by unpaired t-test with categorical data analysed with Fisher’s exact test. Analysis of both the signalling arrays and the individual gene qPCR was carried out using Sabiosciences PCR array data analysis web portal[23 ]. To determine genes of interest, volcano plots were used comparing each of the three groups to each other for both amnion and chorion. Students t test was used to test for differential expression when comparing one group to another. A false discovery rate (p) threshold of 0.1 in conjunction with a fold change threshold of 2 to assign gene significance in the array analysis stage[24 (link)]. This is to screen the large amount of data generated and hone in on genes of interest. The same test and analysis software was used for the qPCR validation but here we used a threshold of p<0.05 to infer statistical significance. Correlation between gestational age and gene expression was estimated by least squares linear regression modelling using a significance of 0.05. Correlation between staging of inflammation (maternal and fetal) and gene expression was also estimated using least squares linear regression modelling; a p< 0.05 was used to define statistical significance.

To examine the range of genes modulated by the expression of VEGFA and the co-expression of VEGFA and BMP2 in BMSC in the three-dimensional scaffold (in vitro), a custom PCR array (cat no.: 330131; SuperArray Bioscience, Frederick, MD, USA) containing primer pairs for 30 genes related to osteogenesis and angiogenesis was used. Total RNA from three biological replicates (n = 3) of ad-GFP, ad-VEGFA, and ad-BMP2 + VEGFA groups at 3 and 14 days was used for cDNA synthesis. PCR amplification was performed using the following cycling conditions: 95 °C for 10 min, (95 °C for 15 s, and 60 ° C for 1 min) × 40 cycles in ABI Prism Sequence Detector 7900 HT (Applied Biosystems, Foster City, USA). Pre- and post- PCR quality control measures, as recommended by the manufacturer, were strictly followed. PCR array data were analyzed as described previously [27 (link)]. Briefly, threshold cycle (Ct) was used to calculate the 2^–ΔCt value for each gene using PCR Array Data Analysis Web Portal (SABiosciences). 2^–ΔCt values were then exported to microarray data analysis software (J-Express 2012). For statistical analysis, unsupervised hierarchical clustering and one-way analysis of variance (ANOVA) with Bonferroni post hoc analysis were used.