one step primescripttm iii rt qpcr mix kit  (TaKaRa)

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a , Schematic of the experimental timeline. K18-hACE2 mice were intranasally challenged with 50 plaque forming units (PFU) of ancestral SARS-CoV-2 and monitored for up to 90 days post-infection (dpi). Tissues were collected at the indicated time points for viral load analysis, multiplex cytokine profiling, histopathology, and bulk RNA sequencing (n = 3-5 mice per group per time point). b, Time-course quantification of viral RNA burden in whole-brain homogenates using RT-qPCR, presented as viral RNA copies per µg of total RNA. c, Heat map of cytokine and chemokine protein levels in brain lysates at the indicated time points, measured using a multiplex array. The data represent log 2 -transformed concentrations (pg mg ¹ protein). Values

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

Techniques: Infection, Multiplex Assay, Histopathology, RNA Sequencing, Quantitative RT-PCR, Transformation Assay, Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a,b , Body weight change ( a ; n = 12 mice per group) and survival ( b ; n = 13 mice per group) of K18-hACE2 mice infected intranasally with 50 PFU of SARS-CoV-2 compared with mock controls. c–f , Time-course quantification of viral RNA in the lung ( c ), nasal turbinate ( d ), eye ( e ) and testis ( f ) at the indicated time points using RT-qPCR (n = 4 mice per group per time point). Limits of detection (LOD) indicated by the dotted lines. The dashed line indicates the limit of detection; viral RNA in the testis was not detected at any time point. N.D., not detected. Data are the mean ± s.e.m. Statistical significance was determined by two-way ANOVA with repeated measures followed by Sidak’s multiple-comparisons test ( a ), log-rank (Mantel–Cox) test ( b ), and one-way ANOVA with Dunnett’s multiple-comparisons test ( c – e ). The exact P values are indicated; **** P < 0.0001.

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

Techniques: Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a , Time-course quantification of Hcrt mRNA levels in whole-brain lysates by RT–qPCR, expressed as fold change relative to mock controls (n = 4 mice per group). b, Representative low-magnification immunohistochemistry (IHC) image of orexin staining in a mock-infected brain, showing expression restricted to the hypothalamic region. Scale bar, 100 µm. c, Representative IHC images of orexin immunoreactivity in the lateral hypothalamus at the indicated time points. Dashed ovals highlight the orexin neuron field. High-magnification insets (right) show individual neuronal morphology. Scale bars, 100 µm (low magnification), 30 µm (high magnification). d, Double immunofluorescence staining for SARS-CoV-2 nucleocapsid (NP; green) and orexin (red) in the hypothalamus in mice infected with SARS-CoV-2 (2 × 10 4 PFU) at 6 days post-infection (dpi). Nuclei were counterstained with DAPI (blue). White arrows indicate orexin-positive neurones in the NP-negative regions. Scale bar, 25 µm. Representative images in b-d from n=3-5 independent biological replicates are shown. Data represent the mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. The exact P values are indicated.

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

Techniques: Quantitative RT-PCR, Immunohistochemistry, Staining, Infection, Expressing, Double Immunofluorescence Staining

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a, Experimental design. BALB/c mice were intranasally challenged with mouse-adapted SARS- CoV-2 (MA10; 4×10 5 PFU) and monitored for up to 90 days post-infection (dpi); Tissues were collected for viral RNA quantification and histopathology at the indicated time points. b,c, Body weight change ( b ; mock n = 5; MA10 n = 12 mice, per group) and survival ( c ; mock n = 26, MA10 n = 32 mice per group) of MA10-infected mice compared with mock controls. d, Time- course quantification of lung viral RNA by RT–qPCR at the indicated time points (n = 4 mice per group per time point). The dashed line indicates the limit of detection. Data are the mean ± s.e.m. Statistical significance was determined using two-way ANOVA with repeated measures followed by Sidak’s multiple-comparison test ( b ), log-rank (Mantel–Cox) test ( c ) and one-way ANOVA with Dunnett’s multiple-comparisons test ( d ). The exact P values are indicated or denoted as * P < 0.05, ** P < 0.01. N.D., not detected.

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

Techniques: Infection, Histopathology, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a , Schematic of the experimental design. C57BL/6 mice were intranasally challenged with the influenza A virus (IAV; PR8 strain, 2 × 10 2 PFU). b,c, Body weight change ( b ; Mock, n = 5; IAV, n = 15) and survival analysis ( c ; Mock n = 5, IAV n = 27) of IAV-infected mice compared with mock controls. d, Time-course quantification of viral RNA loads in the lung at indicated time points using RT–qPCR (n = 4 mice per group). Data represent the mean ± s.e.m. Statistical significance was determined using two-way ANOVA with repeated measures followed by Sidak’s multiple comparison test ( b ), log-rank (Mantel–Cox) test ( c ) or one-way ANOVA with Tukey’s multiple comparison test ( d ). The exact P values are mentioned, or indicated by asterisks as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. N.D., not detected.

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

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

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a , Schematic of the experimental timeline. Human iPSC-derived glutamatergic neurones were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1. Cells were harvested at 72 h post-infection (hpi) for analysis. b, Quantification of intracellular SARS-CoV-2 viral RNA loads using RT–qPCR at 72 hpi. Data are presented as viral RNA copies per µg of total RNA (n = 3 independent biological replicates). c, Western blot analysis of NeuN and SARS-CoV-2 Nucleocapsid (NP) protein expression in mock and SARS-CoV-2-infected neurones at 72 hpi. β- Actin was used as a loading control. Representative immunoblots are shown. Data represent the mean ± s.e.m.

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

Techniques: Derivative Assay, Infection, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a , Schematic illustration of the in vitro experimental design used to assess the specificity of orexin signalling. Human iPSC-derived glutamatergic neurones were pre-treated with the dual orexin receptor antagonist suvorexant (10 µM) or vehicle for 2 h, followed by treatment with recombinant orexin-A/B peptides (γORX-A/B; 5 μM each) at the indicated concentrations; the cells were then harvested at 72 h post-treatment (hpt) for immunoblotting. b, Western blot analysis showing the effect of suvorexant pretreatment on γORX-A/B-mediated NeuN induction. Representative blots (top) and quantification of NeuN protein levels normalized to β-actin (bottom) are shown. The fold change relative to the mock control is indicated below the blots. c, Dose-dependent effects of orexin on NeuN expression. Neurones were treated with increasing concentrations of γORX-A/B (0, 10, and 100 nM) and analysed by western blotting at 72 h post-treatment (hpt). d, Schematic representation of the in vivo rescue experiments. BALB/c mice were intranasally infected with MA10 SARS-CoV-2 (4 × 10 4 PFU) and administered γORX-A/B (2 mg kg ¹) or vehicle daily from 0 to 5 days post-infection (dpi). Brain tissues were collected at 6 dpi. e,f, Quantification of viral burden ( e ) and Hcrt mRNA levels ( f ) in whole-brain lysates at 6 dpi using RT–qPCR. g, ELISA quantification of the orexin-A/B peptide concentrations in brain homogenates. h, Representative Western blots of NeuN expression in whole-brain lysates from mock, vehicle-treated and γORX-A/B-treated mice at 6 dpi. i, Densitometric quantification of NeuN protein levels normalised to β-actin. Data in e – g and i represent mean ± s.e.m. (n = 4 mice per group). Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison test ( f , g , i ) or a two-tailed unpaired Student’s t-test ( e ). The exact P values are indicated. N.D., not detected.

Article Snippet: Quantitative RT-PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using either the One-Step PrimeScript III RT-qPCR Mix (RR600A) or the One Step TB Green PrimeScript RT-PCR Kit II (RR086A) (Takara, Kyoto, Japan), following the manufacturer’s protocols.

Techniques: In Vitro, Derivative Assay, Recombinant, Western Blot, Control, Expressing, In Vivo, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test