Sensimix plus sybr kit

Manufactured by Meridian Bioscience

Sourced in Germany

The Sensimix Plus SYBR Kit is a reagent kit designed for real-time PCR (qPCR) analysis. The kit contains all the necessary components, including a SYBR Green-based master mix, required to perform quantitative gene expression studies.

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Lab products found in correlation

Tetro cdna synthesis kit, by Meridian Bioscience (4 mentions) Trisure reagent, by Meridian Bioscience (4 mentions) Abi 7500 real time pcr system, by Thermo Fisher Scientific (4 mentions) Sybr green, by Meridian Bioscience (3 mentions) Turbo dna free kit, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)

6 protocols using sensimix plus sybr kit

Quantitative RT-PCR Analysis of Hippocampal Neurons

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Total RNA was extracted from primary hippocampal neurons with TRIzol (Invitrogen) according to the manufacturer's instructions. RNA quantitation was performed via quantitative real-time PCR (RT-PCR). The total RNA was treated using the TURBO DNA-free™ Kit (Ambion), reverse-transcribed with SuperScript III reverse transcriptase (Invitrogen), and amplified by using the SensiMixPlus SYBR Kit (BioLine) and the 7900HT Fast Real-Time PCR System (Applied Biosystems). Oligonucleotides amplifying the TATA binding protein (TBP), APP, and RanBP9 were chosen from the Roche Universal Probe Library and were as follows:
Relative changes in gene expression were quantified by applying the comparative threshold method (Ct) after determining the Ct values for the reference gene (TBP, the endogenous control) and the target genes in each sample set according to the 2^−ΔΔCt method. All reactions were performed in triplicate.

Barbato C., Pezzola S., Caggiano C., Antonelli M., Frisone P., Ciotti M.T, & Ruberti F. (2014). A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons. Frontiers in Cellular Neuroscience, 8, 37.

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Quantification of Cytokine-Induced Gene Expression

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B16.F10 melanoma cells were cultured in the presence or absence of IL-33 (100 ng/mL) for 4 h. Total RNA was extracted from tumor cells by using TRIsure reagent (Bioline, London, UK). mRNA was reverse transcribed by means of Tetro cDNA Synthesis Kit (Bioline). Quantitative reverse transcription-PCR (qPCR) with forward and reverse primers for CCL5, CCL24 and HPRT (Eurofins Genomics, Ebersberg, Germany) [6 (link)] was performed using Sensimix Plus SYBR Kit containing the fluorescent dye SYBR Green (Bioline) and by means of an ABI 7500 Real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, Ma, USA). Triplicates were performed for each experimental point. Data were normalized to HPRT (2-ΔCt method) and presented as fold change expression vs. control.

Andreone S., Spadaro F., Buccione C., Mancini J., Tinari A., Sestili P., Gambardella A.R., Lucarini V., Ziccheddu G., Parolini I., Zanetti C., D’Urso M.T., De Ninno A., Businaro L., Afferni C., Mattei F, & Schiavoni G. (2019). IL-33 Promotes CD11b/CD18-Mediated Adhesion of Eosinophils to Cancer Cells and Synapse-Polarized Degranulation Leading to Tumor Cell Killing. Cancers, 11(11), 1664.

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Quantitative Analysis of Immune Gene Expression

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Total RNA was extracted from MCA205-Tlr-3^+/+ or Tlr-3^-/- and from EG.7-OVA tumor lesions by using TRIsure reagent (Bioline, London, UK). The mRNA was reverse transcribed by means of Tetro cDNA Synthesis Kit (Bioline). Quantitative reverse transcription-PCR (qPCR) with forward and reverse primers (see Table 1 for sequence) for TLR-3 (MCA205 only) and IFNγ, IL-2, IL-4, IL-13, IL-17, IL-33, perforin, granzyme B and HPRT (Eurofins Genomics, Ebersberg, Germany) [20 (link)] was performed using Sensimix Plus SYBR Kit containing the fluorescent dye SYBR Green (Bioline) and by means of an ABI 7500 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, Ma, USA). Triplicates were performed for each experimental point. Data were normalized to HPRT (2-ΔCt method) and presented as fold change expression versus. control. A Heatmap of the gene expression was generated using R software [21 ] and its Pheatmap package [22 ].

Rossi A., Lucarini V., Macchia I., Sestili P., Buccione C., Donati S., Ciccolella M., Sistigu A., D’Urso M.T., Pacca A.M., Cardarelli E., Mattei F., Proietti E., Schiavoni G, & Bracci L. (2020). Tumor-Intrinsic or Drug-Induced Immunogenicity Dictates the Therapeutic Success of the PD1/PDL Axis Blockade. Cells, 9(4), 940.

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Quantitative gene expression analysis

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Total RNA was extracted from mBaso by using TRIsure reagent (Bioline, London, UK). Messenger RNA was reverse transcribed by means of Tetro cDNA Synthesis Kit (Bioline). Quantitative reverse transcription-PCR (qPCR) was performed using Sensimix Plus SYBR Kit containing the fluorescent dye SYBR Green (Bioline) and by means of an ABI 7500 Real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Forward and reverse primers (Table SI) were purchased from Eurofin Genomics (Ebersberg, Germany). Triplicates were performed for each experimental point and data were normalized to HPRT (2-ΔCt method).

Gambardella A.R., Poto R., Tirelli V., Schroeder J.T., Marone G., Mattei F., Varricchi G, & Schiavoni G. (2022). Differential Effects of Alarmins on Human and Mouse Basophils. Frontiers in Immunology, 13, 894163.

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Quantifying Immune Responses in Mouse Tumor Models

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Total RNA was extracted from smLN, ingLN and blood of C57Bl/6 mice implanted with B16-OVA and treated with Sl-IFN, Sl-mock and IFNpti for 18 h by using TRIsure reagent (Bioline). mRNA was reverse transcribed by using Tetro cDNA Synthesis Kit (Bioline). Quantitative reverse transcription-PCR (qPCR) with forward and reverse primers for Mx1 and HPRT [44 (link)] (Eurofins Genomics) was performed by SYBR Green technology (Sensimix Plus SYBR Kit) (Bioline) by means of an ABI 7500 Real-time PCR system (Applied Biosystems, Thermo Fisher Scientific) and the following reaction conditions: 15 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C (46 cycles). Triplicates were performed for each experimental point. Data were normalized to HPRT (2-ΔCt method).

Ciccolella M., Andreone S., Mancini J., Sestili P., Negri D., Pacca A.M., D’Urso M.T., Macchia D., Canese R., Pang K., SaiYing Ko T., Decadt Y., Schiavoni G., Mattei F., Belardelli F., Aricò E, & Bracci L. (2021). Anticancer Effects of Sublingual Type I IFN in Combination with Chemotherapy in Implantable and Spontaneous Tumor Models. Cells, 10(4), 845.

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Quantitative Real-Time PCR of β-Actin

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Quantitative real-time PCR was performed in a BioRad CFX96™ real-time PCR by using the SensiMix Plus™ SYBR-Kit (Bioline). Each 20 μ l reaction contained 10 μ l SensiMix-Plus, 1 μ l 50 mM MgCl₂ (final conc. 2.5 mM), 0.25 μ l of a 10 pmol/μ l primer solution using the β-actin primers: ACT 512-F (5' ATG TGC AAG GCC GGT TTC GC 3') and ACT 783-R (5' TAC GAG TCC TTC TGG CCC AT 3') (Carbone and Kohn, 1999 (link)), 6.5 μ l H₂O and 2 μ l of DNA template. The amplification conditions were 95°C for 10 min and then 40 cycles of 95°C 15 s, 61°C 20 s and 72°C 15 s. Fluorescence measurements were made at the end of each annealing cycle and an additional measuring point at 80°C (for 1 s) to detect the formation of primer dimers during amplification. A melt curve analysis was made by raising the temperature from 65 to 95°C in 0.5°C steps for 5 s each.

Ettenauer J., Piñar G., Tafer H, & Sterflinger K. (2014). Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene. Frontiers in Microbiology, 5, 262.

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