Qscript microrna quantification system

Manufactured by Quanta Biosciences

Sourced in United States

The QScript microRNA Quantification System is a laboratory instrument designed for the accurate quantification of microRNA levels. It utilizes a proprietary technology to enable sensitive and specific detection of microRNA targets in a variety of sample types.

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Lab products found in correlation

Perfecta sybr green supermix, by Quanta Biosciences (3 mentions) Qscript microrna cdna synthesis kit, by Quanta Biosciences (2 mentions) Qscript cdna synthesis kit, by Quanta Biosciences (2 mentions) Cfx connect real time pcr detection system, by Bio-Rad (2 mentions) Qscript synthesis kit, by Quanta Biosciences (1 mentions) Cfx connect qpcr detection system, by Bio-Rad (1 mentions) Applied biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions) Ribozol rna extraction reagent, by Avantor (1 mentions) Qscript cdna supermix reagent, by Quanta Biosciences (1 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)

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QScript system

5 protocols using qscript microrna quantification system

Quantitative Real-Time PCR Analysis

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cDNA was made with qScript™ Synthesis kit (Quanta BioSciences Inc.,Gaithersburg, MD). ARPC2 was used as a reference gene for normalization. All real-time PCR (qPCR) reactions were performed in triplicate using PerfeCTa® SYBR® Green SuperMix (Quanta BioSciences) and quantified using the ddCT method. The primer sequences are listed in Table 1.
The qScript™ microRNA Quantification System (Quanta BioSciences, Inc.) was used for miR expression using cDNA generated by the qScript™ microRNA cDNA Synthesis Kit. 50 pg of initial RNA/PCR reaction was used on a CFX Connect™ qPCR Detection System (Bio-rad, Hercules, CA). SNORD48 was used as a reference gene to normalize miR expression.

Ramirez H.A., Pastar I., Jozic I., Stojadinovic O., Stone R.C., Ojeh N., Gil J., Davis S.C., Kirsner R.S, & Tomic-Canic M. (2017). STAPHYLOCOCCUS AUREUS TRIGGERS INDUCTION OF MIR-15B-5P TO DIMINISH DNA REPAIR AND DE-REGULATE INFLAMMATORY RESPONSE IN DIABETIC FOOT ULCERS. The Journal of investigative dermatology, 138(5), 1187-1196.

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RNA Extraction and Quantification Protocol

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RNA from MCs was extracted using Ribozol RNA Extraction Reagent (Amresco) as per the manufacturer’s recommendation, with 1 μg of RNA reverse transcribed into cDNA using qScript cDNA SuperMix Reagent (Quanta Biosciences). miRNA-enriched cDNA was generated using the qScript microRNA Quantification System (Quanta Biosciences). Quantitative real-time PCR was carried out using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). mRNA and miRNA expression and fold changes were calculated using the ΔΔC_T method, where 18S was used as a control for mRNA and U6 snRNA as a control for miRNA. Primers sequences used in the study are provided in Supplementary table 5.

Mehta N., Li R., Zhang D., Soomro A., He J., Zhang I., MacDonald M., Gao B, & Krepinsky J.C. (2021). miR299a-5p promotes renal fibrosis by suppressing the antifibrotic actions of follistatin. Scientific Reports, 11, 88.

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AGO2 Immunoprecipitation and qRT-PCR

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AGO2 immunoprecipitation was performed according to Peritz et al. (54 (link)). AGO2 was precipitated using primary antibody (sc-32877, Santa Cruz, TX, USA, 1:200), followed by qRT-PCR using qScript microRNA quantification system (Quanta Biosciences, MD, USA).

Hanin G., Shenhar-Tsarfaty S., Yayon N., Hoe Y.Y., Bennett E.R., Sklan E.H., Rao D.C., Rankinen T., Bouchard C., Geifman-Shochat S., Shifman S., Greenberg D.S, & Soreq H. (2014). Competing targets of microRNA-608 affect anxiety and hypertension. Human Molecular Genetics, 23(17), 4569-4580.

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Quantitative Analysis of miRNA and Gene Expression

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The qScript microRNA Quantification System (Quanta BioSciences, Inc., Gaithersburg, MD, USA) was used for microRNA expression. Briefly, cDNA was synthesized from total RNA including the microRNA fraction using the qScript microRNA cDNA Synthesis Kit. 50pg of initial RNA were used per PCR reaction on a CFX Connect Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA). SNORD48 was used as a reference gene to normalize the miR expression. For gene expression, cDNA was made with qScript cDNA Synthesis kit (Quanta BioSciences Inc., Gaithersburg, MD, USA). ARPC2 was used as a reference gene for normalization.
All real-time PCR reactions were done in triplicate using the PerfeCTa SYBR Green SuperMix (Quanta BioSciences) and the relative gene expression was calculated with the ddCT method.

Ramirez H.A., Liang L., Pastar I., Rosa A.M., Stojadinovic O., Zwick T.G., Kirsner R.S., Maione A.G., Garlick J.A, & Tomic-Canic M. (2015). Comparative Genomic, MicroRNA, and Tissue Analyses Reveal Subtle Differences between Non-Diabetic and Diabetic Foot Skin. PLoS ONE, 10(8), e0137133.

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Integrative Analysis of miRNA and mRNA Expressions

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To provide more power for integration of genomic with qPCR data, additional NFFs (n=5) and DFUFs (n=4) were prospectively generated and included in validation of miRNA and mRNA expressions. qScript microRNA Quantification System (Quanta BioSciences, Inc.) was used to assess microRNA expression. Briefly, cDNA was synthesized from total RNA including the microRNA fraction using the qScript microRNA cDNA Synthesis Kit (Quanta BioSciences, Inc.). 1ng of cDNA per PCR reaction was used with a CFX Connect^™ Real-Time PCR Detection System (Bio-Rad). Small nucleolar RNA C/D box 48 (SNORD48) was used as a reference gene to calculate miRNA relative expression.
For gene expression analysis, cDNA was made using qScript^™ cDNA Synthesis kit (Quanta BioSciences, Inc.) and actin related protein 2/3 complex, subunit 2 (ARPC2) was used as a reference gene. All real-time PCR reactions were done in triplicate using PerfeCTa SYBR Green SuperMix (Quanta BioSciences, Inc.). Relative gene expression was calculated using the ddCT method. Sequence of primers used in qPCR are presented in Table 1.

Liang L., Stone R.C., Stojadinovic O., Ramirez H., Pastar I., Maione A.G., Smith A., Yanez V., Veves A., Kirsner R.S., Garlick J.A, & Tomic-Canic M. (2016). Integrative Analysis of miRNA and mRNA Paired Expression Profiling of Primary Fibroblast Derived from Diabetic Foot Ulcers Reveals Multiple Impaired Cellular Functions. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 24(6), 943-953.

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