Rotor gene q equipment

For the FISH assay, small EBV RNAs (EBER1), which are highly expressed in cells with latent infection, were investigated in slides with approximately 5 μm of tissue. For this, the Clark Laboratories™ Kit was used according to the manufacturer’s instructions.
The DNA of formalin-fixed, paraffin-embedded (FFPE) tissues of gastric adenocarcinomas (4–5 slices of 5 μm of tissue) was obtained using a QIAamp DNA FFPE Tissue Kit (QIAGEN, Venlo, Netherlands), and DNA from fresh tissue (200 μg) was extracted using TRIzol^® (Thermo Fisher Scientific, Massachusetts, USA), following the manufacturer’s instructions. Then, the DNA was quantified with the Qubit^® dsDNA BR Assay kit on Qubit^® equipment (Thermo Fisher Scientific, Massachusetts, USA). The qPCR test was performed for the detection of the EBV-EBNA-1 gene region, using the qPCRAlert EBV^® Kit (NANOGEM), on the Rotor-gene Q^® equipment (QIAGEN, Venlo, Netherlands), with initial programming at 50 °C for 2 min for decontamination and 95 °C for 10 min for initial denaturation, followed by 45 cycles of 95 °C for 15 s for annealing and 60 °C for a 1 min extension. A standard curve (with controls with 10⁵, 10⁴, 10³, and 10² copies) was used to verify the efficiency of the reaction, and ultrapure water was used as a negative control.

We used qPCR to quantity the absolute abundance of bacterial and archaeal 16S rRNA genes as well as the key genes involved in N cycle pathways, including denitrification (nirS, nirK, nosZ clade I, and nosZ clade II), N fixation (nifH), dissimilatory nitrate reduction to ammonia (DNRA; nrfA), ammonia oxidation (bacterial amoA, archaeal amoA, comammox amoA), and anammox- and n-damo-specific 16S rRNA genes (Supplementary Fig. 7). The qPCR assays were performed using RotorGene^® Q equipment (Qiagen, Valencia, CA, USA). The qPCR method was performed following ref. ^{34 (link)}. Briefly, the qPCR reactions were performed in 10 μL volume containing 5 μL Maxima SYBR Green Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA), an optimized concentration of forward and reverse primers, 1 μL of template DNA and sterile distilled water. The gene-specific primer sets, optimized primer concentrations and thermal cycling conditions for each target gene are shown in Supplementary Data 8. The quantification data were analyzed with RotorGene Series Software (version 2.0.2; Qiagen, Hilden, Germany) and LinRegPCR program (version 2020.0)^{42 (link)}. The gene abundances were calculated as a mean of fold differences between a sample and each 10-fold standard dilution in respective standard as recommended by ref. ^{42 (link)}; gene abundances were reported as gene copy numbers per gram of dry soil.

RT-qPCR amplification assays for swabs and saliva were carried out using reagents from AgPath-ID™ One-Step RT-PCR (Thermo Fisher Scientific, Waltham, Massachusetts, USA). A set of probe-associated primers (assay) aimed at SARS-CoV-2 nucleocapsid (N1 and N2) and envelope (E) genes was used [18 ,19 (link)]. Endogenous control Ribonuclease P/MRP Subunit P30 (RPP30) was also included into the assay to assess sample integrity. The RT-qPCR mixture contained 10 µM of sense and antisense primers, 5 µM of probe, 2X RT-PCR buffer, 25X RT-PCR Enzyme mix (ArrayScript™ Reverse Transcriptase and AmpliTaq Gold^® polymerase), and 5 µL of viral RNA extracted from swabs and 7 µL from saliva. The conditions for the RT-qPCR at Rotor-Gene Q equipment (QIAGEN) were as follows: 45 °C for 15 min for reverse transcription followed by enzyme activation at 95 °C for 10 min, and then 45 cycles were conducted at 95 °C for 15 s and at 55 °C for 45 s. Ct values below 40 for two of the three genes represented positive results. Fluorescence readings were performed using the FAM channel, and the analysis of the results was performed using the Rotor-gene Q series program with the threshold set at 0.1.