Quantinova sybr green kit

Quantitative real-time PCR (QPCR) was performed using the Quantinova SYBR Green Kit (Qiagen, Hilden, Germany), and the fluorescence intensity was measured by StepOne Plus (Applied Biosystem, Foster CA, USA) according to manufacturers’ protocols. Briefly, a reaction master mix was prepared by mixing 10 µL 2x QuantiNova SYBR Green PCR Master Mix, 2 µL of QN ROX reference dye, forward and reverse primer at final concentration of 0.7 µM each, and 50 ng of cDNA per one reaction. The cycling condition was i) PCR initial heat activation at 95 °C for 2 min, ii) 40 cycles of denaturation at 95 °C for 5 s and combined annealing/extension at 60 °C for 10 s, and iii) Melting curve analysis. The final Ct value was normalised to the housekeeping gene and fold changes were analysed using comparative the Ct value to a suitable control. List of primers was listed in Table 2.

qRT-PCR was conducted on RNA extracted from the control and OPMD muscles. For cDNA synthesis, 500 ng RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN) and random primers, following the manufacturer’s instructions. Subsequently, qPCR amplification was performed with the QuantiNova SYBR Green kit (QIAGEN) using 5 ng RNA, with technical duplicates, using a standard amplification protocol at a melting temperature of 60°C. Samples with Ct values above 35 were excluded from the analysis to eliminate potential noise. The average Ct values from the technical duplicates was used for ddCt calculation. PABPN1 Exon-4-5 (ENSG00000100836) was normalized to hypoxanthine ribosyltransferase, and isoforms 201 (ENST00000216727), 202 (ENST00000397276), and 207 (ENST00000556821) to exon 3–4. Isoforms 001 and 004 were targeted with specific primer sets, designed to cover different exons, thereby amplifying only mRNA molecules. Primer sets were designed with the NCBI Primer design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and the primers sequence is in Table S2D.

RNA was extracted from cell pellets with the Qiagen (Hilden, Germany) RNeasy Kit according to the manufacturer´s protocol, including all optional steps. 500 µg of RNA were then transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit from Thermo Fisher Scientific (Schwerte, Germany)according to the manufacturer´s protocol, using Oligo dT primer. To determine expression levels, the QuantiNova SYBR Green Kit from Qiagen (Hilden, Germany)was used according to the manufacturer´s protocol with small changes regarding the composition of the MasterMix: 5 µL SybR Green, 0.05 µL ROX Reference Dye, 2.3 µL H₂O and 0.15 µL primer working solution. 18S served as internal control.
All primers were validated and showed an efficiency between 90 and 110%. The sequences can be found in Table S1.

Genomic DNA (gDNA) was extracted from whole blood using REDExtract-N-Amp Blood PCR Kit (Sigma Aldrich). Total gDNA (proviral load) load was then quantified using Tarlinton et al.^{21 (link)} conserved primers (Table 1) using QuantiNova SYBR green kit (Qiagen) as per manufacturer’s conditions. β-actin PCR was performed in parallel with KoRV PCR as a control for DNA quality and to provide relative copy numbers by normalization using this gene. Standards for β-actin and KoRV of known concentration of 10², 10⁴, 10⁶ and 10⁸ copy number of the target gene sequence^{12 (link)} were prepared as follows. DNA was extracted from a known KoRV positive koala samples and amplified by PCR. The PCR products were electrophoresed in a 2% agarose/TBE (45 mM Tris-borate and 1 mM EDTA, pH 8.0) gel, stained with ethidium bromide (0.5 ug ml⁻¹) and then visualized on a UV transilluminator. The DNA then purified using the High Pure PCR Product Purification Kit (Roche, Applied Science, Germany). Spectrophotometric measurement of absorbance at 260 and 280 nm wavelengths was used to determine the concentration of DNA in the purified preparations. Avogadro’s formula was used to calculate the number of molecules of the product. All reactions were carried out on a Rotor-Gene Q 5-plex HRM platform (Qiagen).

Viral RNA was extracted from the plasma using the Qiagen viral RNA mini kit (Qiagen). Contaminating DNA removal and cDNA synthesis of RNA prepared from plasma was conducted using QuantiTect Reverse Transcription kit (Qiagen). Total cDNA (viral RNA load) load was then quantified using Tarlinton et al.^{21 (link)} conserved primers (Table 1) using QuantiNova SYBR green kit (Qiagen) as per manufacturer’s conditions.
Standards of known concentration of 10², 10⁴, 10⁶ and 10⁸ copy number of the target gene sequence^{12 (link)} were prepared as follows. cDNA was prepared, as above, from a known KoRV positive koala samples and amplified by PCR. The PCR product was electrophoresed in a 2% agarose/TBE (45 mM Tris-borate and 1 mM EDTA, pH 8.0) gel, stained with ethidium bromide (0.5 ug ml⁻¹) and then visualized on a UV transilluminator. The DNA then purified using the High Pure PCR Product Purification Kit (Roche, Applied Science, Germany). Spectrophotometric measurement of absorbance at 260 and 280 nm wavelengths was used to determine the concentration of DNA in the purified preparations. Avogadro’s formula was used to calculate the number of molecules of the product. All reactions were carried out on a Rotor-Gene Q 5-plex HRM platform (Qiagen).

Further investigations were conducted on Shewanella oneidensis because of its relevance as a model organism for studies on extracellular chromate reduction.
Thus, quantitative PCR assays (qPCR) were conducted on amended microcosm tests using the primer sets 640F/815R and Eub338/Eub518, in order to measure the abundance of Shewanella oneidensis during the experiment, in comparison with the total amount of bacteria, until 28 days.
qPCR analyses were performed on a Rotor-Gene Q (Qiagen, Germantown, MD, USA) using the QuantiNova SYBR Green kit (Qiagen). The 20 µL reaction mixtures contained 0.7 µM of each primer, 10 µL of QuantiNova SYBR Green master mix, 5 µL of DNA diluted template corresponding to 1 ng of total DNA, and 2.2 µL of RNase-free water. The conditions were as follows: 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C for denaturation, 30 s at 52 °C for annealing, and 30 s at 60 °C for elongation. Relative quantitation was carried out using all of the bacteria to normalize S. oneidensis abundance, and the concentration at day 0 as the calibrator according to the 2^−ΔΔCt Method [28 (link)].