Type-specific viral load assays were developed in-house to quantify the amount of HPV genomic copies in the DNA isolates for 11 hrHPV types (16/18/33/35/39/45/51/52/56/58/59), potential hrHPV66, and low-risk (lr) HPV6 and HPV11 [16 ,17 (link)]. Due to technical difficulties, the HPV31 viral load assay was excluded from this research. The time between initial HPV genotyping and viral load quantification was approximately 2–9 years. Each viral load assay contains HPV type-specific primers and probes that bind a region of the major capsid protein encoding L1 gene. Briefly, 5 μl total DNA isolate was suspended in 15 μl mastermix containing LightCycler 480 Probes Master (Roche), 400 nM forward primer, 400 nM reverse primer, and 100 nM probe. The viral load assays were carried out on the Roche LightCycler 480 platform (Roche) with cycling conditions of 95 °C for 10 min, 50 cycli of 95 °C for 15 s, and 60 °C for 30 s. The amount of HPV genomic copies per reaction (c/rxn) was corrected for cellular content with a β-actin qPCR to obtain the viral load, which is defined as copies per cell (c/cell). Samples that initially tested positive for HPV, but which were negative in the viral load and/or β-actin tests were excluded from further analyses.
Lightcycler 480 platform
Manufactured by Roche
Sourced in Switzerland, United States, Germany, China
The LightCycler 480 platform is a real-time PCR instrument designed for a wide range of applications, including gene expression analysis, genotyping, and DNA and RNA quantification. The platform utilizes advanced optical and thermal technology to provide reliable and accurate results. It is suitable for use in research laboratories and clinical settings.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr green 1 master mix, by Roche (3 mentions)
Lightcycler 480 sybr green 1 master, by Roche (3 mentions)
Lightcycler 480 probes master, by Roche (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Universal methylated human dna standard, by Zymo Research (2 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (2 mentions)
Sybr green realtime pcr master mix, by Toyobo (2 mentions)
Cdna rt premix kit, by Bioneer (2 mentions)
Primescript rt reagent kit ver 2, by Takara Bio (2 mentions)
Sensifast cdna synthesis kit, by Meridian Bioscience (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Sybr green 1 master kit, by Roche (2 mentions)
Lightcycler 480 high resolution melting master, by Roche (1 mentions)
Sybr green 1 kit, by Roche (1 mentions)
Sybr green master mix, by RiboBio (1 mentions)
66 protocols using lightcycler 480 platform
1
Quantifying HPV Viral Loads Across Genotypes
2
Developing HRM Data Analysis Approach
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To generate HRM data for the development of our data analysis approach, we used four EpiMelt assays, targeting the APC, BRCA1, H19, and MGMT genes (provided by MethylDetect ApS, Aalborg, Denmark). A reference range of controls with different methylation levels was generated. The provided assays included a methylated and non-methylated control. They were mixed to gain a dilution range of 0, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% of the methylated control in a non-methylated background. PCR amplification was performed using LightCycler® 480 High Resolution Melting Master (catalogue number: 04909631001) and Roche LightCycler480 platform (Roche Applied Science, Laval, PQ, Canada). Briefly, the PCR amplification mix contained: 3 mM MgCl2, 1 x LightCycler® High Resolution Melting Master (Roche), 500 nM of the primer mix, and 6 μL of the provided control mix in a total volume of 20 μL. All the reactions were run in triplicates.
3
Quantifying Streptococcus Competence Induction
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Cultures of SV35-T23 were grown in Columbia broth to an OD600 of 0.05. Cultures were induced with CSP2 at a concentration of 0.125 µg/ml. Samples (5 ml) were removed and placed into RNAprotect at three time points, pre-CSP, 8 min post-CSP, and 13 min post-CSP. RNA was extracted from samples with Qiagen RNeasy, and cDNA was synthesized with the Roche Transcriptor first-strand cDNA synthesis kit. Quantitative PCR was done with the Roche SYBR green I kit on the Roche LightCycler 480 platform. The data were analyzed with linregPCR, which uses arbitrary fluorescence units to represent the amount of RNA in each sample (65 (link), 66 (link)). Data were normalized to the expression level of the 16S rRNA gene and represented as the average expression of test genes in replicates, where the error bars indicate the standard deviation of the expression values within replicates (n = 3).
4
Quantification of HPV-45 and HPV-59 Viral Loads
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The viral copy number (VCn) of all DNA samples, previously isolated with the MP96, was quantified with separate HPV-45 and HPV-59 type-specific (TS) real-time quantitative PCR (qPCR) assays. TS qPCR assays for HPV-45 and -59 were developed in previous research (11 (link), 12 (link)). Each TS qPCR assay targets a region on the capsid-encoding L1 gene and is adjusted for specificity from Seaman et al. (13 (link)) and is optimized to exceed sensitivity levels of the SPF10 method. For HPV-45 and -59, primers and probes were designed to target a 107-bp and a 116-bp region within the L1 gene of HPV-45 and -59, respectively (12 (link)). Briefly, 5 μl DNA was added to 15 μl master mix containing LightCycler 480 Probes Master (Roche), 400 nM forward primer, 400 nM reverse primer, and 100 nM probe. The TS qPCR assay was performed on the Roche LightCycler 480 platform (Roche) with cycling conditions of 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 30 s. The specificity and sensitivity of the TS qPCRs for HPV-45 and -59 were determined previously as described by van der Weele et al. (12 (link)). Table S1 in the supplemental material shows the differences between the SPF10 method and the HPV-45 and -59 TS qPCR assays.
5
Quantification of miRNA Expression by RT-qPCR
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Reverse transcriptase quantitative PCR (RT-qPCR) was performed using Bulge-LoopTM miRNA RT-qPCR Starter Kit (RiboBio Co., Ltd, Guangzhou, China) to measure the expression of the target miRNAs. Briefly, miRNA was polyadenylated and reverse-transcribed to cDNA. qPCR amplification was conducted in 96-well plates containing the cDNA products, SYBR Green master mix, and specific primers (RiboBio Co., Ltd, Guangzhou, China). The amplification reactions were carried out on a Roche LightCycler 480 platform (Roche, USA). The expression levels of the miRNAs were normalized to those of cel-miR-39 by the ΔΔCt method using the following formula: ΔΔCt=Ct[miRNA of interest] Ct[cel-miR-39]. The relative expression levels of miRNAs in patients were obtained following the 2-ΔΔCt method.
6
Quantitative HPV Typing and Viral Load
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Samples were tested for HPV DNA using the highly sensitive SPF10-PCR DEIA/LiPA25 system (version 1). If tested positive for HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and/or 59, we determined the HPV VL by using a previously described quantitative type-specific (q)PCR targeting the L1 region, optimized to approach SPF10-LiPA25 sensitivity levels [16 (link),17 ]. HPV VLs were corrected for the number of human cells in each sample and expressed as genomes per human cell [16 (link)]. qPCRs were performed in 20 μl final volume using LightCycler TaqMan Master on the Roche LightCycler 480 platform (Roche Diagnostics, Almere, the Netherlands). The lower limit of detection varied for each HPV type, ranging from 200 to 920 copies/ml [16 (link),17 ]. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 were defined as high-risk HPV (hrHPV) and types 6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 68/73, 70 and 74 as low-risk HPV (lrHPV). HPV VL was categorized in three groups: (1) hrHPV and HPV 6/11 negative, (2) hrHPV and/or HPV 6/11 positive with an undetectable VL, and (3) hrHPV and/or HPV 6/11 positive with a detectable VL. This classification was made to distinguish between possible HPV deposition (group 2) and actual HPV infection (group 3).
7
Quantitative Analysis of Transcription Factors
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The total RNA was extracted using the TRIzol reagent (RR047A; Takara, Japan). The cDNA was synthesized and used as the template for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) performed using the SYBR Green Premix Pro Taq HS qPCR Kit (cat. no. AG11701; Accurate Biology). The qPCRs for Sox9, surfactant protein c gene (Sftpc), and epithelial cell adhesion molecule gene (Epcam) in technical triplicates and using three 10-fold dilutions of standardized genome equivalents were performed using the SuperScript III One-Step RT-PCR Kit (12574026; Thermo Fisher Scientific, Waltham, MA, USA) and the Roche LightCycler 480 platform. RT-qPCR was performed using an Eppendorf Realplex 4 instrument (Eppendorf, Hamburg, Germany). The sequences of the PCR primers were obtained from Univ-bio (Shanghai, China) and are provided in Table S1 . The relative expression of each gene was normalized to that of Actb.
8
RNA extraction and qRT-PCR protocol
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Total RNA was extracted from tissues or cell lines using TRIzol reagent (15596018, Thermo) and standard protocols were followed. Subsequently, cDNAs were synthesized using the PrimeScript™RT kit (R232-01, Vazyme). The Roche LightCycler 480 platform (Roche, GER) was employed to quantify gene expression levels, utilizing SYBR qPCR Master Mix (Q111-02, Vazyme). Supplementary Table 1
9
Investigating NMD Inhibition Impact on GFP
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GFP reporters were agroinfiltrated with the p14 silencing suppressor and either mock or the dominant-negative Upf1 inhibitor of NMD as previously described [25 (link)]. At 5 days post-infiltration, leaves were imaged and collected for RNA extraction using Trizol. Total RNA was treated with RQ1 DNase (Promega) prior to reverse-transcription quantitative PCR (RT-qPCR). Small PCR fragments (<200 bp) were amplified using the SYBR Green-based Luna One-Step RT-qPCR kit according to the manufacturer’s protocol (New England BioLabs). The Roche LightCycler 480 platform was used for all experiments. p14 served as an internal reference gene for relative GFP gene expression using the 2−∆∆Ct method.
10
Quantifying Transporter Expression in Tissues
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Snap-frozen tissue samples were ground to a powder, and total RNA was extracted by resuspending in Tri-Reagent (Thermo Fisher Scientific) and processing using standard procedures. Following determination of the concentration and purity of isolated RNA by A260/A280 spectrophotometry, cDNA was prepared from 3 µg of total RNA in a total volume of 20 µl using the Roche Transcriptor First Strand cDNA Synthesis kit (Roche, Burgess Hill, UK).
Relative quantification of gene expression was undertaken for seven transporters (ABCB1, ABCG2, ABCC2, SLCO2B1, SLC51A, SLC51B, and CDH-17) by real-time polymerase chain reaction using the Roche Universal Probe Library (UPL) system (Roche). Using the UPL Genefinder software, gene-specific intron-spanning primers and appropriate fluorescent hydrolysis probes were designed for each transporter. Assays were performed using the Roche Lightcycler 480 platform in a total volume of 20 µl with 200 nM forward and reverse primers, 100 nM of the UPL hydrolysis probe, and ∼0.3 µg cDNA. The comparative threshold cycle method was used to determine mRNA expression relative to reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and villin 1. Sequences of the polymerase chain reaction primers are supplied inSupplemental Table 4 .
Relative quantification of gene expression was undertaken for seven transporters (ABCB1, ABCG2, ABCC2, SLCO2B1, SLC51A, SLC51B, and CDH-17) by real-time polymerase chain reaction using the Roche Universal Probe Library (UPL) system (Roche). Using the UPL Genefinder software, gene-specific intron-spanning primers and appropriate fluorescent hydrolysis probes were designed for each transporter. Assays were performed using the Roche Lightcycler 480 platform in a total volume of 20 µl with 200 nM forward and reverse primers, 100 nM of the UPL hydrolysis probe, and ∼0.3 µg cDNA. The comparative threshold cycle method was used to determine mRNA expression relative to reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and villin 1. Sequences of the polymerase chain reaction primers are supplied in
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