Ssofast evagreen supermixes

The relative expression pattern of silicon transporter genes viz. EcLsi1, EcLsi2, and EcLsi6 and defense hormone regulating genes viz. SAM (Ethylene), LOX (Salicylic acid), and PAL (Jasmonic acid) were analyzed using qPCR (CFX96TM Real-Time System, Bio-Rad, USA) using SSO Fast Eva Green Supermixes (BioRad, USA) following manufacturer’s instruction. The housekeeping gene, EcActin, was used as an internal control for normalization of data obtained. Reaction conditions for thermal cycling were 95 °C for 60 s, followed by 40 cycles of 95 °C for 20 s, 60 °C for 15 s, then 72 °C for 30 s. Fluorescence data were collected during the cycle at 60 °C. The melting curve was used as an internal check for assessing quality of the gene amplification. The experiments were repeated twice independently with three replicates each time. The threshold cycles (Ct) were used to find the relative expression level of all the genes over control using the comparative 2^−ΔΔCt method^{62 (link)}. Gene specific primers used in this study are listed in Table S6 and S7.

The number of copies of BcoAT genes was determined using quantitative polymerase chain reaction (qPCR), using the methods described by Louis et al. [13 (link)] with modifications. The PCR reaction was performed in a total volume of 10 µL using the SsoFast EvaGreen^® Supermixes (Bio-rad, Hercules, CA, USA) with 500 nM of each of the forward [GCNGANCATTTCACNTGGAAYWSNTGGCAYATG] and reverse primers [CCTGCCTTTGCAATRTCNACRAANGC] and 1 µL of DNA samples. Amplification and detection of DNA by real-time PCR were performed with the BioRad CFX384 Real Time System in duplicate. The reaction conditions for amplification were 98 °C for 2 min, 35 cycles of 98 °C for 10 s, 60 °C for 45 s, 72 °C for 45 s.

The abdominal aorta was harvested, cleaned of perivascular adipose tissue, flash frozen in liquid nitrogen, and stored at −80ºC. RNA was extracted using an QIAgen RNEASY kit as per manufacturer guidelines, and cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. Quantitative PCR was performed using SsoFast EvaGreen Supermixes (Bio-Rad, Hercules, CA, USA) and Bio-Rad CFX™ Real Time system. The 2^−ΔΔCt was used to quantify relative gene expression. Primer sequences are provided in Supplementary Table 1.

Whole cell RNA was extracted from HEK 293 cells grown in 6-well plates using TRIzol. Residual DNA was removed using the TURBO DNA-free kit (Life Technologies). First strand cDNA synthesis was performed with 1 μg RNA using M-MuLV reverse transcriptase (NEB), following manufacturer’s instructions. The sample was subsequently digested with RNaseH (NEB). qPCR was performed with SsoFast EvaGreen Supermixes (BIO-RAD), using 0.5 μM β-actin or MICU1 primers, and 0.5, 2.5, 5, or 10 ng RNA for producing a standard curve. Detection of the PCR product was done with a CFX96 real-time PCR detection system (BIO-RAD), using the following protocol: 95°C for 30 s, 50 cycles of 95°C for 5 s and 57°C for 5 s. The sequence of the primers is provided in Supplementary file 1.
ΔCt was calculated by subtracting the Ct for β-actin from the Ct for MICU1, with 3 independent RNA extractions and qPCR measurements using 2.5 ng whole RNA. ΔΔCt was calculated by subtracting the mean ΔCt for control WT cells from the mean ΔCt for each stable MICU1 knockdown cells. The results were presented as the percentage of MICU1 mRNA in MICU1 knockdown cells relative to MICU1 mRNA in WT control, using the equation% mRNA = 1/2^|ΔΔCt|.

Total mRNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. mRNA was converted into cDNA using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's protocol. Quantitative PCR was performed on 96‐well plates using SsoFast EvaGreen Supermixes (Bio‐Rad) with the Bio‐Rad CFX™ Real‐Time System. Gene expression was normalized to 18s, and fold change was calculated using the 2^−ΔΔCt method. Primer sequences are provided in the Table S3.

Briefly, DNA from mice tails was collected via swabbing the challenge site with buccal swabs and extracted as described in http://home.ccr.cancer.gov/lco/VirionExtraction.htm. PCR reaction assays were performed with primers forward 5’-GGTCAAAAGGGCAGCGTCTA-3’, reverse 5’-TGCTTCCCCTCTTCCGTTTT-3’ and were run using SsoFast EvaGreen Supermixes (Bio-Rad) according to the manufacturer’s protocol. Primers were assessed for specificity and sensitivity (S3 Fig). Analysis of MusPV E1^E4 mRNA transcript by reverse transcriptase quantitative PCR (RT-qPCR) was performed as described in [33 (link)]. Briefly, mouse tail tissue specimens were flash frozen and stored at -80°C. Tissues were disrupted using a frozen mortar and pestle and homogenized with QIAshredders (Qiagen). RNA for qRT-PCR was extracted using RNeasy Mini kit (Qiagen). cDNA was generated using iScript Advanced cDNA Synthesis Kit (Bio-Rad) and run using TaqMan Gene Expression Master Mix (Applied Biosystems). Primer sequences were E1^E4-forward, 5′-CATTCGAGTCACTGCTTCTGC-3′; E1^E4-reverse, 5′-GATGCAGGTTTGTCGTTCTCC-3′; E1^E4-probe, 5′-6-carboxyfluorescein (FAM)-TGGAAAACGATAAAGCTCCTCCTCAGCC-6-carboxytetramethylrhodamine (TAMRA)-3′. Beta-actin primers (Life Technologies) was used to normalize DNA collection. Triplicate reactions were performed for each primer-probe set.