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Quanta qscript cdna supermix

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Sourced in United States, France

Quanta qScript cDNA SuperMix is a ready-to-use reagent for reverse transcription of RNA to cDNA. It contains all the necessary components for efficient conversion of RNA to cDNA in a single tube.

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Lab products found in correlation

Power sybr green master mix 2x, by Thermo Fisher Scientific (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Nanodrop 1000, by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Lightcycler 480 sybr green 1 master, by Roche (1 mentions) Rneasy kit, by Qiagen (1 mentions) Icycler, by Bio-Rad (1 mentions) Lightcycler 480, by Roche (1 mentions)

5 protocols using quanta qscript cdna supermix

1

Analyzing Gene Expression in MS Patients

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The expression of LDLRAP1, N4BP2L2, PGAP3, SNX2, ABHD8, AIM2, ANXA1, DDX54, EAF2, FASN, RHOT1, SNPH genes was analyzed by RT–qPCR using the SYBR Green technology (LightCycler® 480 SYBR Green I Master, Roche). ACTB was used as a housekeeping gene. The sequences of the different primers are listed in Annex S1. Experiments were run on a light cycler 480 thermocycler and each reaction was done in triplicate. The relative gene expression was calculated as follows: Cttarget/CtACTB. The University Hospital Bern cohort included leftover RNA from the RNA sequencing experiment and consisted of 10 relapsing GC‐sensitive and 15 relapsing GC‐resistant patients with MS. In addition, samples from 21 relapsing GC‐sensitive and one relapsing GC‐resistant patients with MS from the Eginition University Hospital Athens were added to the cohort. RNA extraction was done as described in previous sections. The quantity and quality of the extracted RNA were assessed using a NanoDrop microvolume spectrophotometer (Witec AG). cDNA was prepared from 100 ng RNA mixed with Quanta qScript cDNA SuperMix (VWR International). Reverse transcription was done using a thermal program of 5 min 25°C, 30 min 42°C, and 5 min 85°C. RNA was stored at −80°C.
Bagnoud M., Remlinger J., Joly S., Massy M., Salmen A., Chan A., Karathanassis D., Evangelopoulos M, & Hoepner R. (2023). Predicting glucocorticoid resistance in multiple sclerosis relapse via a whole blood transcriptomic analysis. CNS Neuroscience & Therapeutics, 30(2), e14484.
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2

RNA Extraction and qPCR Analysis

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Organ pieces are immediately placed in RNAlater during dissection and stored at 4°C until RNA is extracted using RNeasy Mini Kit (Qiagen) and following manufacturer’s instructions. The isolated RNA is quantitated and either stored at −80° in preparation for RNA-seq or 1μg RNA is converted to cDNA using Quanta qScript cDNA supermix from VWR. Quantitative PCR using a reaction mix containing 1:40 dilution of each cDNA, 10 μM paired primers listed in Data S1 and Power-SYBR Green 2x Master Mix (Applied Biosystems) is performed on an Applied Biosystems Step One Plus Real-Time PCR system. The program is run for 40 cycles at 60o. The relative gene expression when normalized to GAPDH is calculated using the ddCt algorithm.
Resnick-Silverman L., Zhou R., Campbell M.J., Leibling I., Parsons R, & Manfredi J.J. (2023). In vivo RNA-seq and ChIP-seq analyses show an obligatory role for the C terminus of p53 in conferring tissue-specific radiation sensitivity. Cell reports, 42(3), 112216.
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3

RNA Extraction and qPCR Analysis

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Organ pieces are immediately placed in RNAlater during dissection and stored at 4°C until RNA is extracted using RNeasy Mini Kit (Qiagen) and following manufacturer’s instructions. The isolated RNA is quantitated and either stored at −80° in preparation for RNA-seq or 1μg RNA is converted to cDNA using Quanta qScript cDNA supermix from VWR. Quantitative PCR using a reaction mix containing 1:40 dilution of each cDNA, 10 μM paired primers listed in Data S1 and Power-SYBR Green 2x Master Mix (Applied Biosystems) is performed on an Applied Biosystems Step One Plus Real-Time PCR system. The program is run for 40 cycles at 60o. The relative gene expression when normalized to GAPDH is calculated using the ddCt algorithm.
Resnick-Silverman L., Zhou R., Campbell M.J., Leibling I., Parsons R, & Manfredi J.J. (2023). In vivo RNA-seq and ChIP-seq analyses show an obligatory role for the C terminus of p53 in conferring tissue-specific radiation sensitivity. Cell reports, 42(3), 112216.
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4

RNA Isolation and cDNA Synthesis from Murine Tissues

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Splenic-derived CD3+ T cells, spinal cords and brains were isolated from control (CD3+ T cells and spinal cords: n = 5; brains: n = 9) or SP-treated (CD3+ T cells and spinal cords: n = 5; brains: n = 9; 30 mg/kg/day) mice at peak of MOG35-55 EAE disease corresponding to day 4 after treatment initiation (for disease course and body weight of the mice see Figure A1 and Figure A2). RNA was extracted following manufacturer’s instructions (RNeasy Kit, Qiagen, Hilden, Germany). Complementary DNA (cDNA) was prepared from 1 µg RNA mixed with Quanta qScript cDNA SuperMix (VWR International, Radnor, PA, USA). Reverse transcription was done using a thermal program of 5 min 25 °C, 30 min 42 °C and 5 min 85 °C. RNA was stored at −80 °C.
Bagnoud M., Briner M., Remlinger J., Meli I., Schuetz S., Pistor M., Salmen A., Chan A, & Hoepner R. (2020). c-Jun N-Terminal Kinase as a Therapeutic Target in Experimental Autoimmune Encephalomyelitis. Cells, 9(10), 2154.
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5

Quantitative RT-PCR Analysis of Viral mRNA

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B6C3H brains were dissected and RNA was extracted from brain regions using RNeasy lipid tissue Minikit (ref. 74804, Qiagen, Courtaboeuf, France). RNA samples were tittered using Nanodrop (Nanodrop 1000, Thermo Fisher scientific, Villebon sur Yvette, France). 500 ng of RNA were reverse-transcripted with Quanta qScript cDNA Super Mix (ref. 95048-100, VWR international, Fontenay-sous-Bois, France) on a Biorad iCycler. After a 1:10 dilution, complementary DNA samples were analyzed by qPCR using primers specific for the WPRE region of viral mRNA, and primers specific for GAPDH mRNA. We used the LC 480 SYBR Green 1 Master kit (ref. 001,352,04887, Roche, Boulogne-Billancourt, France) on a Roche Light Cycler 480 to perform the qPCR and analyzed the results on the LC 480 software.
Sargent D., Bétemps D., Drouyer M., Verchere J., Gaillard D., Arsac J.N., Lakhdar L., Salvetti A, & Baron T. (2018). Investigating the neuroprotective effect of AAV-mediated β-synuclein overexpression in a transgenic model of synucleinopathy. Scientific Reports, 8, 17563.
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