Taqman universal master mix 2 with ung

For RT-PCR analysis, total RNA was isolated with a Total RNA Kit II (OMEGA, BioTek, D6934-01) in RNase-free conditions from cells lysed in RNA lysis buffer and then reverse transcribed into cDNA with RNA reverse transcriptase kit (TaKaRa, RR037A) according to the manufacturer’s protocol. RT-PCR was then performed with a TB Green PCR kit (TaKaRa, RR820A) using the ABI QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) to determine the expression levels of the genes of interest. The mouse organ tissues were homogenized to detect the virus gene copy number by qPCR (TaqMan™ Universal Master Mix II with UNG, 4440044, Applied Biosystems). All primers were synthesized by the Beijing Genomics Institute (Shenzhen, China). The primer sequences are listed in Supplementary Table 1.

The cDNA was obtained from serum using the RT (Reverse Transciption) kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). Pre-designed commercial assay of TaqMan probes from Thermo Fisher Scientific were specific to miR-22 (hsa-miR-22-3p ID 000398). The expression level of miRNA was evaluated using the comparative threshold cycle method (ΔCt) and was normalized with a corresponding sequence of C. elegans miRNA as an exogenous normalizer in gene expression (spike-in cel-miR-39). The relative concentration of miR-22 was described as a fold change (2-ΔCt), by using the equation ΔCt = (Ct miRNA-Ct spike). The cut-off value was established as cycle ≤40, and it was considered that a gene was not detectable when Ct >40 and the signal was below the established limits [16 (link),17 (link)].

RNA was extracted from whole ovaries or tumour pieces using RNeasy Plus mini extraction kit (Qiagen, Germany) and reverse transcription performed using QuantiTect Reverse transcription kit (Qiagen, Germany). All quantitative real time rtPCR assays were carried out three times using TaqMan® universal master mix II with UNG (Applied Biosystems, USA), Taqman® assays (Additional file 1: Table S3) and QuantStudio™ 7 Flex Real Time PCR system (ThermoFisher, USA), and relative expression levels determined using QuantStudio™ 7 Real Time PCR software.

A One‐Step RT‐PCR Kit (Applied Biosystems, USA) was used to amplify HMPV RNA. Each 20‐μL reaction mixture comprised 10 μL of 2× RT‐PCR buffer, 0.4 μL of forward and reverse primers (20 μM), 0.4 μL of probe (10 μM), 0.8 μL of 25× RT‐PCR reverse transcriptase enzyme, 4 μL of template RNA, and 4 μL of ddH₂O. The real‐time PCR conditions were 50°C for 30 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. TaqMan Universal Master Mix II with UNG (Applied Biosystems) was used to detect HBoV. Each 20‐μL reaction mixture comprised 10 μL of buffer, 0.4 μL of forward and reverse primers (20 μM), 0.4 μL of probe (10 μM), 0.8 μL of 25× RT‐PCR reverse transcriptase enzyme, 4 μL of template RNA, and 2.8 μL of ddH₂O. The real‐time PCR conditions were 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds (Table 1). To standardize quantification of HBoV and HMPV, known numbers of DNA or RNA transcripts containing the primer targets were used in 10‐fold serial dilutions (10⁰ to 10⁷ copies/μL). Any amplification detected before 40 cycles was considered positive. Quantification of >10⁰ copies/μL (10³ copies/mL) was considered a positive result. An internal positive control gene (GAPDH), positive control, and negative (water) control were included in all reactions.

SNP genotyping was done using Restriction Fragment Length Polymorphism (RFLP) for 10/13 SNPs. For the remaining three SNPs, genotyping was done using TaqMan as no restriction site for a single cutter restriction enzyme was identified either for the wild type or variant allele. For both genotyping methods, 10% of the genotyping results were confirmed to be true using Sanger Sequencing. SNP genotyping using RFLP was done for Cyp1A1m1, Cyp1A2*F, GSTP1, NAT2, CDKN1A, CDKN1B, CDKN2A, RET L769L, S836S and S904S polymorphisms and using TaqMan for CDKN2B, CDKN2C and RET G691S polymorphisms. For RFLP, 100 ng gDNA was PCR amplified followed by restriction digestion using reaction conditions as per the manufacturer's protocol. The digested products were visualized on 2% agarose gel and the genotypes were inferred from band sizes in the gel. For TaqMan SNP genotyping, 1 µL gDNA (10 ng/µL) was mixed with 2.5 µL TaqMan universal master mix II with UNG (Applied Biosystems, cat#4440038) and 0.1 µL probe mix (Applied Biosystems) designed for each SNP. TaqMan realtime PCR was performed on QuantStudio 5.0 and genotypes were inferred from amplification plot and allelic discrimination plots. About 5% of all the genotyping results were validated using Sanger Sequencing.

The expression of selected transcripts was evaluated by retrotranscription and quantitative PCR (RT-qPCR) by using TaqMan probe-based assays (Applied Biosystems, Foster City, CA, USA). Total RNA extraction and retrotranscription were performed as described in the Microarray processing section. PCR was performed in a 20 μL total volume reaction by mixing 100 ng cDNA, 2X TaqMan Universal Master Mix II with UNG (Applied Biosystems, Foster City, CA, USA), 1μL of specific target TaqMan assay probe, and 1 μL of primer limited probe for the endogenous control glyceraldehyde-3-phosphate deshydrogenase (GAPDH). Amplification was conducted at 50 °C for 2 min, 95 °C for 10 min as initial step followed by 95 °C for 15 sec, 60 °C for 1min for 40 cycles on a StepOne Real Time PCR System (Applied Byosistems, Foster City, CA, USA). Relative gene expression was calculated using the 2^ΔΔCT method.