Quantstudio 5 real time pcr instrument

Messenger RNA (mRNA) was extracted from each CTC line using the RNeasy Mini Kit (74106, QIAGEN, Hilden, Germany), and complementary DNA (cDNA) was obtained by reverse transcription (RT) using the SuperScript III First-Strand Synthesis Super Mix kit (18080, INVITROGEN, Carlsbad, USA), according to the manufacturer’s instructions. Semi-quantitative PCR that targeted a wide range of sequences (Supplementary Table S2) was used to assess the expression of a large panel of genes, including all the transcripts previously validated as up- or down-regulated after a microarray assay in the CTC-MCC-41 line^{14 (link)}. In all experiments, β2-microglobulin (B2M) was used as reference housekeeping gene. One µL of each RT product was added to 9 µL of mix containing the two primers (6 µM each) and SYBR Green (600882, Brilliant III Ultra-Fast SYBR Green Master Mix, AGILENT TECHNOLOGIES, Santa Clara, USA). PCR reactions were performed on a QuantStudio 5 real-time PCR instrument (THERMO FISHER, Waltham, USA) as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and then 1 cycle at 95 °C for 1 min, 55 °C for 5 s, 95 °C for 30 s, 37 °C for 30 s. Results were analyzed using the QuantStudio Design and Analysis Software. Hierarchical clustering was computed with the Cluster and Treeview software packages (PMID: 9843981).

Total RNA was extracted from tongue cancer patient samples using AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, cat. no. 80224) as per the manufacturer’s protocol. Extracted RNA was resolved on 1.2% agarose gel to confirm the RNA integrity. DNase treatment was done using a DNA-free™ kit (Ambion, Foster City, CA, USA; cat. no. AM1906). For analyzing transcript levels of F. nucleatum and miRNA target genes, cDNA was synthesized with 500 ng of total RNA using high-capacity cDNA reverse transcription kit (Applied Biosystems, cat. no. 4368814). For miRNA quantification, cDNA synthesis was carried out with 500 ng of total RNA using pooled miRNA-specific stem–loop RT primers [protocol as described (37 )]. Quantitative real-time PCR was performed on QuantStudio5 Real-Time PCR instrument (Thermo Fisher Scientific) using Mir-X miRNA qRT-PCR SYBR Master Mix (Clontech Takara, cat. no. 639676) for miRNAs and KAPA SYBR Fast Universal Mix (KAPA Biosystems, cat. no. KK4601) for Fusobacterium and miRNA target genes. Expression of candidate miRNAs and genes was calculated as ΔCT. RNU48 was used as an internal control for microRNAs and beta-actin for Fusobacterium and genes.

To evaluate the protective effect of the recombinant virus rPRVXJ-delgE/gI/TK-S, viral loads of PRV and PDCoV in blood and anal swabs were measured. The viral DNA/RNA was purified from a 1 g tissue sample using the Magnetic Pathogen DNA/RNA Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The qPCR test was conducted at a total volume of 20.0 µL containing 10.0 µL 2× TaqMan Fast Advanced Master Mix (ThermoFisher, Massachusetts, USA), 2.0 µL DNA, 6.5 µL sterilized H₂O, and 0.5 µL each of the primers (final primer concentration 0.5 mM). The reaction was heated to 95℃ for 1 min, followed by 40 cycles of 95℃ for 5 s, and 60℃ for 20 s (fluorescence captured). TaqMan real-time PCR was carried out using the QuantStudio 5 real-time PCR instrument (ThermoFisher, Massachusetts, America). According to the CT value of each tissue sample, the original copies were calculated by the standard curve constructed in the previous study [15 (link), 23 (link)] and converted into copies of the virus load in each gram of tissue with multiples (25×). The copy numbers of each tissue sample were expressed as log10 copies per gram of tissue sample.