Peptidylprolyl Isomerase

PCR reactions were performed in 96-well plates with an ABI PRISM^® 7500 Sequence Detection System (Applied Biosystems) using SYBR^® Green to detect dsDNA synthesis. Reactions were done in 25 μl volumes containing 200 nM of each primer, 5 μl cDNA (corresponding to ~3 ng), and 12.5 μl 2× SYBR Green Master Mix Reagent (Applied Biosystems). Aliquots from the same cDNA sample were used with all primer sets in each experiment. Reactions were run using the manufacturer's recommended cycling parameters of 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. No-template controls were included for each primer pair and each PCR reaction was completed in triplicate. Dissociation curves for each amplicon were then analyzed to verify the specificity of each amplification reaction; the dissociation curve was obtained by heating the amplicon from 60°C to 95°C (See additional file 1: Dissociation curve data).
Data were analyzed using the SDS 1.2.2 software (Applied Biosystems). Expression levels were determined as the number of amplification cycles needed to reach a fixed threshold in the exponential phase of the PCR reaction (C_t). All amplification plots were analyzed with an Rn threshold of 0.2 to obtain C_t values. The PCR efficiency was determined for each gene with LinReg software, which uses absolute fluorescence data captured during the exponential phase of amplification of each reaction [23 (link)]. Results from the SDS and LinReg software were imported into Microsoft Excel for further analyses and to correct for the different PCR efficiencies [24 (link)]. All primer pairs had efficiencies higher than 1.80 with the exception of EF1-α (m) (1.70) and β-tubulin (1.54). Each was run on the full pericarp developmental series for 2003 and 2004. In addition, all primer pairs except those targeting β-tubulin and cyclophilin were run on 2003 mesocarp samples.
In order to evaluate reference gene stability among samples, three statistical approaches were incorporated. In the first approach, C_t difference (C_{t max}-C_{t min}) and CV were calculated for each gene throughout each development series tested. During the second approach, C_t values were converted into relative quantities and imported into geNorm v.3.4 software [16 (link)]. Analyses were performed both with and without EF1-α (m) data to evaluate whether co-regulation with EF1-α biased the results, considering that the EF1-α (m) targets EF1-α as well as other paralogs. Finally, in the third approach, the standard deviation was calculated for each mean C_t difference (C_t-mean Ct) [17 (link)]. β-tubulin, cyclophilin, and EF1-α (m) were excluded from this third approach due to their poor performances during earlier analysis.
To score reference genes based on gene stability, a scoring scheme was implemented whereby results were combined from each statistical approach for each sampling year. Given that cyclophilin, β-tubulin and EF1-α (m) were excluded from the final analyses (Table 3), they could not be included in the final scoring scheme. For all other genes, the results from each statistical approach (CV, M or 2× SD) within a dataset (2003 or 2004 pericarp development series) were distributed and assigned a score value between 1 to 100, an arbitrary scoring range. For example, the CV values for the 2003 samples ranged from 0.98 for SAND to 2.24 for UBQ-L40. SAND was assigned an arbitrary value of 1 and UBQ-L40, an arbitrary value of 100. Then all other genes were assigned values between 1 and 100, scaled based on their relative distribution. Once all scores were derived for each statistical approach, a cumulative score was used to deduce the final standing.

Brains of wild-type and APPPS1 mice previously treated for Tregs depletion or amplification were harvested at 4 months of age after transcardiac perfusion with PBS. Cerebellum was removed and total RNA was extracted from hemi-brains using RNeasy lipid tissue midi kit (Qiagen). Following DNase treatment, RNA quality was verified using an Agilent Bioanalyzer and quantity measured with a Nanodrop 1000 (ThermoFisher Scientific). For cDNA synthesis, 2 µg of total RNA were processed using RT² First Strand kit (Qiagen). Quality control of RNA and cDNA samples was assessed using RT² RNA QC PCR Array (Qiagen). Relative expression of specific mRNAs was assessed by SYBR green-based real-time quantitative PCR using RT² SYBR Green ROX (Qiagen) and LightCycler 96 Instrument (Roche). Specificity of all primers was validated using Primer BLAST database analysis, and PCR efficiency (> 90%) was validated after optimizing the concentration and annealing temperature. Amplification conditions were: 95 °C for 5 min, then 45 cycles at 95 °C for 10 s, 15 s at either 60 °C or 56 °C depending on the primer pairs, and 72 °C for 30 s. A melting curve was generated at the end of amplification cycles for assessing the specificity of the reaction. Hypoxanthine-guanine phosphoribosyltransférase (HPRT) and peptidylprolyl isomerase A (PPIA) were used as reference housekeeping genes for normalization. Relative expression of genes was evaluated as fold changes using the mean of the control group as reference, and was calculated as 2^−ΔΔCt. All primers were ordered from Eurogentec (sequences available in Additional file 8: Table S1). Data were analyzed using LightCycler 96 software (Roche) and the R software environment.

Total RNA was extracted from frozen muscle samples using PureLink™ RNA Mini Kit (Invitrogen Canada, Burlington, ON) or Trizol (Life Technologies) following the manufacturer’s instructions. Quantification and purity of RNA were assessed using the A260/A280 absorption method. Total RNA (2 μg or 400 ng) was reverse transcribed using a Superscript II® Reverse Transcriptase Kit and random primers (Invitrogen Canada, Burlington, ON) or the SupersCript IV (Life technologies). Real‐time PCR detection of mRNA expression was performed using a Prism® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) or a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) with SYBR Green chemistry (Power-UP SYBR®Green PCR Master Mix, Applied Biosystem). Cycle threshold (CT) values were obtained for each target gene. ΔCT values (normalized gene expression) were calculated as CT of the target gene minus CT of the geometric means of three housekeeping genes (Cyclophilin, Gapdh, β-Actin, and/or 18 S), unless otherwise indicated in the figure legends. Relative mRNA level quantifications of target genes were determined using the threshold cycle (ΔΔCT) method, as compared to Ctrl. The primer sequences for all genes are found in Supplementary Table S5.

Expression of VEGFa, PAI1, MMP9, GLUT1, PGK1, and PPIB were determined by reverse transcription of total RNA followed by semi-quantitative real-time PCR analysis on an Applied Biosystems ABI Prism 7000 thermocycler using Power SYBR Green Master Mix following the manufacturer’s protocol as previously described [7 (link), 14 (link), 15 (link)]. We determined the relative levels of gene expression from a single pooled sample made from three individually wells (biological replicates) from a 12-well plate for stable knockdown HCT116 or HT29 cells maintained under the indicated condition. The results of triplicate experiments, with each sample measured as triplicates, were expressed as 2 ^{-(gene-of-interest number of cycles- cyclophilin number of cycles)}.