qpcr mix  (PCR Biosystems Ltd)

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A to C ) HIEs were pretreated for 3 hours with the indicated concentrations of TAK-779 and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the absence or presence of the same concentrations of TAK-779. After washing, the cells were cultured for 48 hours at 37°C in the absence or presence of TAK-779. Viral GEs at 1 and 48 hours postinfection were quantified by RT-qPCR. Mean data compiled from two independent experiments (J2 and J8FUT2-KI HIEs) and three independent experiments (J4FUT2-KI HIEs) with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors. Significance was determined using two-way ANOVA with Tukey’s post hoc test comparing TAK-779 concentrations and individual experimental results at 48 hours postinfection. P values are shown for individual TAK-779 concentrations versus no TAK-779 treatment control (* P value <0.05, ** P < 0.01, *** P < 0.001, and ****, 0.0001). Significant differences between experiments were observed for the studies conducted in J4FUT2-KI and J8FUT2-KI HIEs ( P < 0.0001 for each).

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Control

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: Different conditions, pre- versus post-treatment (indicated) with pretreatment alone compared to post-treatment alone and 24 hours pretreatment, were compared. J4FUT2-KI HIEs were treated as indicated with 30 μM TAK-779. HIEs were then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the indicated conditions. After washing, the cells were cultured for 48 hours at 37 ° C in the presence of 30 μM TAK-779. Viral GEs at 1 and 48 hours postinfection were quantified by RT-qPCR. Mean data compiled from three independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors. Numbers above lines represent log 10 increase between no treatment versus indicated treatments. Significance was determined using two-way ANOVA with post hoc Tukey’s test comparing 48-hour replication, no TAK-779 to each condition of TAK-779 ( P value,**, < 0.001 and ****, 0.0001). hr, hours.

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Cell Culture, Quantitative RT-PCR

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI HIEs were pretreated for 3 hours and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the presence of the indicated concentrations of TAK-779. After washing, the cells were cultured for the indicated times at 37°C in the presence of the indicated concentrations of TAK-779. Virus replication was evaluated by RT-qPCR at the indicated time points. ( B and C ) After 24 and 48 hours postinfection incubation, monolayers were fixed with methanol. GII.3-positive cells (green) were detected using guinea pig anti-HuNoV VLP Ab (green) and nuclei (blue) were detected by DAPI. (B) Representative images from each condition at 48 hours postinfection are shown. Scale bars denote 14 μm. (C) Numbers of HuNoV infected cells (Foci) per well from 24 and 48 hours postinfection condition were counted. Mean data compiled from two experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors using one-way ANOVA with post hoc Newman Keuls test comparing GEs per well for TAK-779 concentrations at 48- and 72-hour time points, ( P value, ****, 0.0001).

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Cell Culture, Virus, Quantitative RT-PCR, Incubation

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI and ( B ) J8FUT2-KI HIEs were pretreated for 3 hours and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) with or without 30 μM TAK-779, labeled as P1. Following infection, both cells and supernatant were collected at 96 hours postinfection, and a portion was used to quantify viral RNA by RT-qPCR. The virus was subsequently used to infect a fresh batch of HIEs for the next passage, continuing for up to five passages. dpi, days postinfection; hpi, hours postinfection.

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Labeling, Quantitative RT-PCR, Virus

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI and J8FUT2-KI HIEs were pretreated and then infected with GII.3 HuNoV (2.9 × 10 5 GEs per well) in the presence of 30 μM TAK-779 and indicated as P1. Postinfection, the cells and supernatant were harvested at 96 hours postinfection; after processing the supernatant, an aliquot was used to quantify the viral RNA by RT-qPCR. The virus was then used to infect a new batch of HIEs for the next passage, continuing up to 10 passages. ( B ) Immunofluorescence staining of HuNoV-infected J4FUT2-KI monolayers at P5 and P2. At 48 hours postinfection, cells were fixed with methanol and stained with guinea pig anti-HuNoV VLP antibody (green), rabbit anti-NTPase (red), and mouse anti-VPg (white). Nuclei were counterstained with DAPI (blue). Representative images are shown at high (10-μm scale bar) and low (20-μm scale bar) magnification. dpi, days postinfection; hpi, hours postinfection.

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Quantitative RT-PCR, Virus, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI HIEs were pretreated for 3 hours with 30 μM TAK-779 and infected with GII.3 HuNoV derived from stool [2.9 × 10 5 genome equivalents (GEs) per well] or from stock 1 (2.58 × 10 5 GEs per well), stock 2 (2.93 × 10 5 GEs per well), stock 3 (1.02 × 10 5 GEs per well), or stock 4 (2.50 × 10 5 GEs per well). Infections were performed in the presence of 30 μM TAK-779 (left) or vehicle control (right). After washing, the cells were cultured for the indicated times at 37°C in the presence (left) or absence (right) of TAK-779. Virus replication was evaluated by RT-qPCR at the indicated time points. Viral replication was assessed by RT-qPCR. The characterization of each stock was performed once with three replicates. Error bars denote SD. ( B ) Representative images of stock-infected HIEs fixed with methanol at 48 hours postinfection. GII.3-positive cells were detected using guinea pig anti-HuNoV VLP Ab (green) and nuclei (blue) were detected by DAPI. Scale bars denote 14 μm. ( C ) Electron micrograph of HuNoV particles from the supernatant of HIEs 24 hours postinfection infected with stock virus. Scale bar, 50 nm; red line, ~40 nm, expected particle diameter.

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Derivative Assay, Control, Cell Culture, Virus, Quantitative RT-PCR

Journal: Science Advances

Article Title: Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids

doi: 10.1126/sciadv.aeb0455

Figure Lengend Snippet: ( A ) J4FUT2-KI HIEs were pretreated with 30 μM TAK-779 and then infected with GII.3 (TCH-04-577/P21, 2.9 × 10 5 GE per well; TCH-25-396/P12, 2.2 × 10 5 GE per well), GII.17 (1295-44/P13, 2.8 × 10 5 GE per well; TCH-25-256/P171, 3.1 × 10 5 GE per well), G1.1 (BCM-723–100595/P1, 3.7 × 10 5 GEs per well; 51899/P1, 6.9 × 10 5 GE per well). ( B ) J4FUT2-KI HIEs were pretreated with 30 μM TAK-779 and then infected with GII.4 Sydney (BCM 16-2/P31, 3.0 × 10 5 GE per well; TCH-24-163/P16, 2.2 × 10 5 GE per well), GII.4 Den Haag (TCH-23-323/P16, 5.6 × 10 6 GE per well), GII.4 Hunter (TCH-05-797/P4, 3.35 × 10 5 GE per well) in the absence or presence of 30 μM TAK-779. After washing, the cells were cultured for 48 hours at 37°C in the absence or presence of 30 μM TAK-779. Virus replication at 1 and 48 hours postinfection was evaluated by RT-qPCR. Mean data compiled from two independent experiments with three wells per experiment are shown; error bars show SD. Experiments are denoted with different symbol colors Error bars denote SD. Significance was determined using paired t test comparing 48-hour replication, 0 to 30 μM TAK-779 ( P value, *, 0.01; **, 0.01; ***, 0.001; and ****, < 0.0001). hr, hours.

Article Snippet: RT-qPCR was performed with qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences) in an Applied Biosystems StepOnePlus thermocycler.

Techniques: Infection, Cell Culture, Virus, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: An AI-guided framework reveals conserved features governing microRNA strand selection

doi: 10.1093/nar/gkaf1510

Figure Lengend Snippet: HiTmiSS assay and analysis on mixed tissue. ( A ) HiTmiSS protocol for RNA extraction, extension, reverse transcription, and quantification in high throughput. Steps include (i) C. elegans growth and homogenization, (ii) RNA extraction and isolation, (iii) RNA polyuridinylation, extension, and reverse transcription (details shown in ii; orange: miRNAs, green: polyU, purple: SL-polyA reverse universal primer), (iv) addition of 5p or 3p miRNA-strand-specific forward primers, (v) loading into 384-well plates, and (vi) quantification. ( B ) Heatmap of miRNA expression ( n = 3) in mixed tissue clustered by quantity, strand use, and developmental stage. Stages include embryo ( E ), larval 1 through 4 (L1-L4), and YA. The heatmap legend represents the log 10 of expression values for each miRNA strand, normalized by the spike-in GFP control, at each stage, ranging from −4 (deep purple) to 4 (bright yellow). Black color indicates instances of data that fall below the strict expression threshold (one standard deviation above the mean of all expression values at that stage). ( C ) Heatmap of pairing frequency by position for the used (left) and discarded (right) strand of the 20 most highly quantified 5p and 3p Singletons in our mixed tissue dataset. (i) blue: paired; red: unpaired). (ii) Average pairing frequency, and logo plot of nucleotide frequency at each position displayed below (gold box marks the seed regions).

Article Snippet: During this homogenization step, anti-GFP magnetic antibody beads (Chromotek GFP-Trap Magnetic Agarose) were prepared by diluting 25 μl bead slurry in 500 μl dilution buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.5 mM EDTA) and rotating end-over-end at 4°C for 3 min.

Techniques: RNA Extraction, Reverse Transcription, High Throughput Screening Assay, Homogenization, Isolation, Expressing, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: The two-step purification method ViREn identifies a single NSUN6-mediated 5-methylcytosine modification promoting dengue virus RNA genome turnover

doi: 10.1093/nar/gkag003

Figure Lengend Snippet: Establishment of ViREn. ( A ) Schematics of the ViREn workflow. DENV RNA from either infected cells or virions was separated by sucrose gradient ultracentrifugation. DENV gRNA and sfRNA were further enriched using antisense affinity capture. The purified DENV gRNA was subjected to RNA sequencing-based approaches for the identification and mapping of m 5 C modifications. The distribution of RNAs extracted from infected cells ( B ) and virions ( C ) across sucrose density gradient fractions was analyzed by denaturing agarose gel and corresponding northern blot using a hybridization probe targeting DENV 3′UTR. The percentage of DENV gRNA in each fraction is shown at the bottom (mean ± SD, n = 3). ( D ) DENV gRNA collected in fraction 11 was further purified by affinity capture. Its integrity was visualized by northern blotting. ( E ) The level of DENV gRNA and 28S rRNA contaminant, before and after affinity capture, was measured by probe-based RT-qPCR (mean ± SD, n = 3). ( F ) The total RNA composition of fraction 11 before and after affinity capture was analyzed by Illumina-based RNA-seq ( n = 3). Shown is the proportion of reads in each sample corresponding to DENV gRNA, human genome, rRNAs, and unmapped reads (mean ± SD, n = 3).

Article Snippet: Briefly, 3 μl of extracted RNA were mixed with 12 μl qPCR mix (qPCRBIO Probe 1-Step Go Lo-ROX, PCR Biosystems) according to the manufacturer instructions and using the following cycling protocol: 50°C 10 min, 95°C 1 min, 95°C 10 s, 60°C 1 min; steps 3–4 were repeated 39 times.

Techniques: Infection, Purification, RNA Sequencing, Agarose Gel Electrophoresis, Northern Blot, Hybridization, Quantitative RT-PCR