Cfx real time pcr machine

Ovules and seeds used for total RNA extraction were frozen in liquid nitrogen immediately after harvest and stored at −80°C prior to extraction. Four independent biological samples were used for each analysis. Each replicate comprised the content in ovules/seeds of 10 to 15 pistil/siliques. Total RNA was extracted using the RNeasy Mini kit (Qiagen), including RNase-Free DNase Set (Qiagen) treatment during washing, according to the manufacturer’s instructions, and subsequently stored at −80°C. The Superscript Reverse Transcriptase II kit (Invitrogen) was used to generate cDNA from 1 µg of total RNA. Each cDNA sample was diluted 1:125 in water. Quantitative PCRs were performed with the SYBR Green kit (Bio-Rad) on a Bio-Rad CFX real-time PCR machine. For each reaction, 4.4 µL of diluted cDNA were added to 5 µl of SYBR Green and to 0.3 µl of each primer (10 µM) (Supplemental Table 2). Melt curves have been performed and primer efficiency has been tested (primers with efficiency between 85% and 100% have been used in this study). Expression levels were first normalized by the geometrical mean of the expression levels of four reference genes (GAPDH, AT4G12590, AT4G02080, and AT3G25800) (Dekkers et al., 2012 (link)), and subsequently normalized by the expression level of the adequate control. Means and standard deviations were calculated from four independent biological samples.

ECs were isolated with antibody-coated beads, as explained above. Instead of plating cells for culture, cells were lysed with RLT buffer and RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, 74034) as per manufacturer's guidelines. Total RNA was converted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) according to kit instructions. Diluted cDNA was used for qPCR performed using SYBR Green Master Mix (ThermoFisher Scientific) with primers for the following genes: Tgfb, Eng, Tie1, Pecam1, Itga5, Flt4, Flt1, Fgfr2, Sox18, Nrp1, Adipoq, Fabp4, Cyp1b1, Lepr, Aldh1a2, TNFα, Il6. β-actin was used as a housekeeping gene.
To confirm the loss of gene function at the transcript level for gene deletion experiments, ECs were isolated from femurs of tamoxifen-injected transgenic mice for qPCR using primers for each gene (Esr1, Esr21, Gper1, Cpt1a). Primer sequences used in this study are provided in a supplementary table 1.
Reactions were run on a CFX real-time PCR machine (Bio-Rad) using CFX manager 3.1 software. Data were calculated using the ΔΔCt method to show fold gene expression or relative mRNA levels.

The PCR reactions were performed using 50 ng of DNA obtained from genomic tissue and 1 µl for DNA solution obtained from blood. The reaction mixture contained 0.4 µM of forward and reverse primers (F: 5′-AGTCGGCTGATCGTTTTCGA-3′; R: 5′-AATTCCTCCAAGCAGCGGATA-3′), 1× Premix Ex Taq (2× premix -Takara System) and 0.3 µl of SYBR Green (from a 100× stock, Invitrogen). For tissue PCR the genomic region encoding the IL12 p40 was utilized as the control to demonstrate equal amounts of purified DNA were used for all PCR amplifications. The genomic IL-12 p40-specific primers utilized were 5′-GTAGAGGTGGACTGGACTCC-3′ and 5′-CAGATGTGAGTGGCTCAGAG-3′[21] (link). PCR was performed using the Bio-Rad CFX real time PCR machine with cycling conditions as follows: 95°C for 30 seconds; 95°C for 5 seconds and 64.2°C for 30 seconds, for 45 cycles. Amplification was immediately followed by a melt analysis to confirm specific amplification of parasite DNA [19] (link), [23] (link), [24] (link).

RNA was isolated from adipocytes using the miRNeasy Mini Kit (Qiagen, Hilden, Germany, #217004). After reverse transcription (QuantiTect Reverse Transcription Kit, Qiagen,#205314), expression levels of UCP1 (fw: 5′-GCCACTCCTCCAGTCGTTA-3′, rev: 5′-TCTCTCAGGATCGGCCTCTAG-3′) and RPS13 (fw: 5′-CTTGTGCAACACCATGTGAA-3′, rev: 5′-CCCCACTTGGTTGAAGTTGA-3′) were analyzed with the CFX real-time PCR machine (Biorad) using SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma, #S4438). Relative expression was analyzed using the ΔCt method.