Cfx manager software version 3

Threshold detection levels were calculated automatically by the Bio-Rad CFX Manager Software version 3.0 (Bio-Rad). When raw data were not normally distributed and variances were not homogeneous, they were log-transformed before analysis. The t-test was used to compare virus quantities measured at different sampling times. Statistical tests were performed with SIGMAPLOT 13 (Systat Software).

Intergenic links and qPCR analysis were performed on cDNA using primers listed in Additional file 8. Intergenic links were analyzed as previously described [45 ]. qPCR was carried out on a CFX96 real-time PCR detection system (Biorad, France) and the software Manager CFX 3.1 using RNA16S gene as a reference for normalization. For qPCR, the reaction volume was 15 μL and the final concentration of each primer was 0.5 μM. The cycling parameters of the qPCR were 98 °C for 2 min, followed by 45 cycles of 98 °C for 5 s, 56 °C for 10 s, 72 °C for 1 s. A final melting curve from 65 to 95 °C is added to determine the specificity of the amplification. To determine the amplification kinetics of each product, the fluorescence derived from the incorporation of EvaGreen into the double-stranded PCR products was measured at the end of each cycle using the SsoFast EvaGreen Supermix 2X Kit (Bio-Rad, France). The results were analyzed using Bio-Rad CFX Manager software, version 3.0 (Bio-Rad, France). For each point, a technical duplicate was performed. The amplification efficiencies for each primer pairs comprised between 80 and 100%.

Two-step qRT-PCR was used to quantitatively measure gene expression using the iScript^TM Reverse Transcription Supermix for RT-qPCR (Bio-Rad, USA) and a CFX96 Touch ^TM Real-Time PCR Detection System with Bio-Rad CFX Manager software version 3.0 (Bio-Rad). All primer pairs were designed using NCBI PrimerBlast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences are shown in Table S1. The amplification was performed for the fliC, katE, katG, tftC, trxA and 16 S rDNA genes in duplicate in 10 µl total volume containing 5 µl of 2 iTaq Universal SYBR Green (Bio-Rad), 1 µl of cDNA, 0.4 µl of each primer (10 µmol/l), and 3.2 µl of distilled water. The cycle conditions were as follows: 1 cycle of 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. After amplification, melting curve analysis was conducted by increasing the annealing temperature by 0.1 °C per step from 65 °C to 95 °C. The 16 S gene was used as the reference for calculating the relative expression levels of other genes. The normalised expression levels were calculated by using the 2^−ΔΔCt method, where ΔΔCt = (Ct _{target gene} − Ct _{16S rDNA gene}) at other temperatures − (Ct _{target gene} − Ct _{16S rDNA gene}) at 37 °C^{43 (link)}.

Quantitative real-time PCR (qRT-PCR) analyses were performed on a CFX96 Real-Time System (Bio-Rad). The reaction volume was 15 μL and the final concentration of each primer was 0.5 μM. The cycling parameters of the qRT-PCR were 98°C for 2 min, followed by 45 cycles of 98°C for 5 s and 60°C for 10 s and a final melting curve from 65°C to 95°C to determine the specificity of the amplification. To determine the amplification kinetics of each product, the fluorescence derived from the incorporation of EvaGreen into the double-stranded PCR products was measured at the end of each cycle using the SsoFast EvaGreen Supermix 2X Kit (Bio-Rad, France). The results were analyzed using Bio-Rad CFX Manager software, version 3.0 (Bio-Rad, France). The RNA16S gene (Dv16SA) was used as a reference for normalization. For each point a technical duplicate was performed. The amplification efficiencies for each primer pairs were comprised between 80 and 100%. All of the primer pairs used for qRT-PCR are reported in Supplementary Table S2.

Quantitative real-time PCR (qPCR) and the corresponding analysis were performed on a CFX96 Real-Time System (Bio-Rad). The reaction volume was 15 μL and the final concentration of each primer was 0.5 μM. The cycling parameters of the qRT-PCR were 98°C for 2 min, followed by 45 cycles of 98°C for 5 s, 55°C for 10 s, 72°C for 1s. A final melting curve from 65°C to 95°C was performed to determine the specificity of the amplification. To determine the amplification kinetics of each product, the fluorescence derived from the incorporation of EvaGreen into the double-stranded PCR products was measured at the end of each cycle using the SsoFast EvaGreen Supermix 2X Kit (Bio-Rad, France). The results were analyzed using the Bio-Rad CFX Manager software, version 3.0 (Bio-Rad, France). The 16S RNA gene was used as a reference for normalization. For each point a technical duplicate was performed. The amplification efficiencies for each primer pairs were comprised between 80 and 100%. All of the primer pairs used for qRT-PCR are reported in the S4 Table.

Hepatocyte lysates were collected, after transfection and apo(a)/Lp(a) treatment, using RLT Plus buffer (Qiagen) supplemented with 1% (v/v) 2-mercaptoethanol. Total RNA was isolated from the lysates using the RNeasy Plus kit (Qiagen) as per the manufacturer’s protocol. LRP-8, LRP-1 or VLDLR mRNA expression was assessed by qRT-PCR utilizing primer pairs A/B, C/D, E/F, respectively (Table 1) and normalized to GAPDH using primer pair G/H (Table 1). All DNA primers utilized in this study were synthesized by Integrated DNA Technologies, Inc. Reactions were assembled in triplicate using the iTaq Universal SYBR Green One-Step kit (Bio-Rad) according to the manufacturer’s protocol, and performed on a Bio-Rad CFX Connect. Relative quantification of the respective mRNA was determined by normalization of the threshold cycle for each sample with that of GAPDH (primer set G/H; Table 1) from the same reaction well using Bio-Rad CFX Manager software version 3.1 (Bio-Rad). All mRNA expression data are reported as a fold-increase compared with the control sample from cells that were transfected with the pCMV6 parental plasmid.