Rotor gene q series software 2

Antigen detection for RSV in NPA was done by IFA (D3 Ultra Respiratory Virus Screening and ID Kit—Diagnostic Hybrids, United States—Catalog No: 01-010000.v2) along with seven viruses: RSV, influenza-A (Inf-A), Inf-B, adenovirus, para influenza-1 (PIV-1), PIV-2, and PIV-3. Fluorescence microscopy for IFA was done under Leitz Diaplan and Zeiss Axio-cam fluorescent microscopes, Germany for the detection of cells expressing specific antigens for these seven viruses with positive and negative controls. Detailed findings on Inf-A, Inf-B and PIV-1, PIV-2 and PIV-3 are presented in two research articles published from the same program (Rafeek et al., 2021 (link), 2022 (link)).
Nucleic acid extraction from IFA positive samples for RSV was conducted using QIAamp Viral RNA Mini Kit (Qiagen, Germany, Catalog No: 52906). Extracted RNA were tested by a real-time reverse transcription polymerase chain reaction (rtRT-PCR) for RSV subtyping (RealStar^® RSV RT-PCR Kit 3.0 Altona Diagnostics, Germany, Catalog No: 193013), using the Rotor-Gene 6,000 real time PCR machine and Rotor-Gene Q Series Software 2.3.1 (Corbett Life Science, Australia).

The primers of nine RGs (Table 2) were chosen due to their different biological functions and irrelevance. Metabolism-related genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M); ribosomal genes: ribosomal protein large P0 (RPLP0); cellular structure and cytoskeleton: beta-actin (ACTB); transcription and translation: eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), TATA box binding protein (TBP); cell signaling and regulation: peptidylprolyl isomerase A (PPIA), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ); nucleotide metabolism: hypoxanthine phosphoribosyltransferase 1 (HPRT1). GAPDH, B2M, EEF1A1, RPLP0, PPIA, and YWHAZ were referenced from a previous report [35 (link),38 (link)]. ACTB, TBP, and HPRT1 were designed using the PrimerQuest™ tool at an annealing temperature of 60 °C and analyzed using OligoAnalyzer 3.1, a software that confirms the absence of hairpins, homodimers, and heterodimers. To evaluate primer efficiency, RT-qPCR was performed with the 10-fold diluted cDNA of the SF-MSCs, following a previously established protocol [38 (link)]. Standard curve parameters were calculated using the RotorGene Q Series software 2.1.0 (Qiagen, Hilden, Germany).

The relative mRNA expression levels of pluripotent markers (NANOG and OCT4) and apoptotic markers (BAK and BAX) were analyzed by qRT-PCR in triplicates. Total RNA from DP-MSCs treated with or without 10 μM S1P (Cayman chemical, Ann Arbor, MI, USA) in a culture medium for 1 h was extracted using the easy-spinTM Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, South Korea) according to the manufacturer’s instructions. The RNA was quantified using an OPTIZEN NANO Q spectrophotometer (Mecasys, Daejeon, South Korea), and cDNA was synthesized with 500 ng of RNA using HiSenScriptTM RH(-) RT PreMix Kit (iNtRON Biotechnology). qRT-PCR was performed using RealMODTM Green AP 5x qPCR mix (iNtRON Biotechnology) and specific primers for the genes. The PCR reaction cycle consisted of an initial activation at 95 °C for 12 min, followed by 40 cycles of PCR at 95 °C for 15 s, 60 °C for 25 s, and 72 °C for 25 s. The amplification curves, melting curves, and cycle threshold values (Ct values) were analyzed using the Rotor-Gene Q Series Software 2.1.0 (Qiagen, Helden, Germany). The Ct values were normalized to the expression level of the housekeeping gene, ACTB, and all samples were analyzed in triplicate to ensure reproducibility. The relative mRNA expression levels were calculated using the 2^−ΔΔCt method. The primers used in this study are listed in Table 1.

HRM data were generated with the Rotor-Gene™ 6000 and the Rotor-Gene Q Series Software 2.1.0 (Qiagen, Germany).
The final reaction volume of 10 μl contained 1× HRM mastermix (HOT FIREPol® EvaGreen® HRM Mix, Solis BioDyne, Estonia), 150 nM of the forward and reverse primer each, and 2 ng genomic DNA. All samples and ‘no template controls’ (NTC) were analysed in duplicates.
The HRM analysis with pre-amplification was performed with an initial phase of 14 min at 95 °C, 45 cycles of 95 °C/ annealing temperature depending on the primer pair (Online Resource 1)/72 °C for 10 s/ 20 s/ 20 s. After PCR, a hold of 95 °C/1 min was implemented for complete denaturing of all nucleotide strands. HRM was done with a ramp of ~ 10 °C around the melting point of the synthesized DNA fragment (Online Resource 1) and in increments of 0.1 °C and 1 s hold before the temperature increase.

In order to determine the load of calpox virus in various organs and body fluids, a real-time PCR targeting the gene region of the ankyrin repeat-containing protein (GenBank accession number HQ420898) was used. A quantified molecular DNA standard with the target DNA cloned into a TOPO-TA vector (Invitrogen, Karlsruhe, Germany) was applied to calculate the concentration of calpox viral DNA either per gram of tissue or per microliter of blood. The TaqMan^® Universal PCR Master Mix (Thermo Fisher Scientific (formerly Life Technologies), Darmstadt, Germany) was used as follows: 7.5 µL of TaqMan^® Universal PCR Master Mix was mixed with 1 µL of each primer at a concentration of 10 nmol (Calp1for: 5′-CCggCATgCgTgACTgAATT-3′ and Calp1rev 5′-TAAgATgCgAgCCgAgAAgC-3′), 0.5 µL of the 10 nmol of TaqMan probe TIB-1 (FAM -TgCTCCgTgTTCTACCATCgTgCg-TAMRA), 4 µL molecular biology grade water and 1 µL of the extracted DNA. Oligonucleotides were synthesized by TIB Molbiol (Berlin, Germany). The real-time PCR was performed using the 5-Plex Rotor-Gene Q (Qiagen, Hilden, Germany) and the following cycling conditions were applied: initial activation at 95 °C for 10 min, and 45 cycles of 94 °C for 15 s, and 58 °C for 45 s. Absolute quantification was conducted via Rotor-Gene Q Series Software 2.1.0 (Qiagen, Hilden, Germany).

Individuals (genotyped using conventional PCR) were classified into five groups based on genotype type (3/3, 4/4, 3/4, 4/5, 5/5) and the results were tested by qPCR-HRM. PCR master mixtures contained 100 ng of genomic DNA (all gDNA samples were quantified using The Quant-iT^TM dsDNA Assay Kit; Life Technologies, Carlsbad, CA, USA), 250 nM of each primer, 4 μl of 5x AmpliQ HOT EvaGreen HRM Mix (Novazym, Poznań, Poland) and nuclease-free water (AppliChem GmbH, Darmstadt, Germany) in a total volume of 20 μl. The qPCR-HRM analysis was performed a Rotor-Gene Q instrument (QIAGEN GmbH, Hilden, Germany). The qPCR-HRM thermal profile was as follows: 15 min at 95°C for initial denaturation, subsequent 40 cycles (denaturation at 95°C for 15 s, primer annealing at 61°C for 20 s and product synthesis at 72°C for 20 s) and final elongation at 72°C for 1 min for complete product extension. Final elongation was then followed by pre-hold temperatures − 95°C for 15 s and 50°C for 15 s for product re-association and heteroduplex formation. Fluorescence data acquisition (melting curves) was performed during the melting phase (temperature change from 70 to 95°C at a transition rate of 0.1°C each step). High resolution melting curve analysis was performed using the Rotor-Gene Q Series Software 2.3.1 (QIAGEN GmbH, Hilden, Germany).

The amplification and melting curves obtained by PCR-HRM were assessed and exported using Rotor-Gene Q Series Software 2.3.1 (Qiagen). The PSQ data were evaluated and exported with PyroMark Q24 Advanced software 3.0.0 (Qiagen). The exported data were analyzed and are presented graphically using R version 3.6.2 [37 ]. The R-packages used, including corrplot, ggplot2, polynom, rstatix, survival, and survminer, are listed in the Supplementary File R-packages.
For HRM analysis, derivative melting curves were calculated from normalized melting curves by applying Savitzky–Golay filtering for third-degree polynomials. DNA methylation levels obtained by PSQ ≤ 5.00% (lower limit of quantification, LLOQ) and ≥95.00% (upper limit of quantification, ULOQ) were substituted with default values, namely 2.50% and 97.50%, respectively [38 ].
One-way ANOVA (analysis of variance) followed by a post hoc t-test corrected for multiple testing by Holm’s p-value adjustment was applied to test for significant differences between the groups. Groups consisting of only one member were excluded from testing. A scatterplot and Pearson´s correlation coefficient were used to assess the relationship between two quantitative variables. A Kaplan–Meier estimator with a log-rank test was used to analyze the survival data. p-values ≤ 0.05 were considered statistically significant.