Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions, followed by cDNA synthesis of 1μg total RNA with Quanta qScript™ cDNA Synthesis Kit (Quanta Biosciences). iQ SYBR Green Supermix (Bio-Rad) was used for quantification of mRNA, which was performed on a Bio-Rad CFX96 Real-Time PCR Detection system. CFX Manager software was used to determine the crossing points for incorporation of the SYBR Green. Relative expression was determined with β-actin as a reference gene. Primers are as follows: Batf 5′-AGCTTCAGCCGCTCTCCT-3′ and 5′-GGTGTCGGCTTTCTGTGTCT-3′, Irf4 5′-ACGCTGCCCTCTTCAAGG-3′ and 5′-GCTCCTCTCGACCAATTCCT-3′, p21 5′-TCCACAGCGATATCCAGACA-3′ and 5′-GCGCAACTGCTCACTGTC-3′, CyclinD2 5′-GCTAGGAACATGCACACTGC-3′ and 5′-CTGGGGCTTCACAGAGTTGT-3′, γ1-GLT: 5′-GGCCCTTCCAGATCTTTGAG-3′ and 5′-GGATCCAGAGTTCCAGGTCACT-3′, γ3-GLT: 5′-AACTACTGCTACCACCACCACCAG and 5′-ACCAAGGGATAGACAGATGGGG-3′, μ-GLT: 5′-CTCTGGCCCTGCTTATTGTTG-3′ and 5′-GAAGACATTTGGGAAGGACTGACT-3. Primers for AID, β-actin, μ-chain, γ1-chain and γ3 have been previously described (17 (link)).
Quanta qscript cdna synthesis kit
Manufactured by Quanta Biosciences
Sourced in United States
The Quanta qScript™ cDNA Synthesis Kit is a reverse transcription reagent used to convert RNA into complementary DNA (cDNA). It contains a high-performance reverse transcriptase enzyme and components for efficient first-strand cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Isogen Life Science (1 mentions)
Sybr green method, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
4 protocols using quanta qscript cdna synthesis kit
1
Gene Expression Profiling with qRT-PCR
2
Quantitative Real-Time PCR Protocol
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cDNA was generated from 1 μg total RNA using a Quanta qScript cDNA Synthesis kit (Quanta BioSciences, MD, United States). rpsL was used as housekeeping gene control and the primers were listed in Table 2 . The qPCR was performed using PowerUpTM SYBRTM Green Master Mix (Thermo Fisher Scientific) on an Eco Illumina real-time detection system (Montreal Biotech) under the following conditions: UDG activation at 50°C for 2 min, Dual-LockTM DNA polymerase initiation at 95°C for 2 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Fold changes of gene expression levels were calculated according to the 2–ΔΔCq method. Each sample was measured in triplicate and repeated at least three times.
3
RNA Extraction and cDNA Synthesis
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RNA was extracted from 200 μL of frozen whole blood using Macherey-Nagel Nucleospin RNA Blood Kit (Bethlehem, PA) with DNase digestion and eluted with 60 μL nuclease-free water. 15 μL of RNA was used for cDNA synthesis with Quanta qScript cDNA Synthesis Kit (Cat# 95047–100, Quanta Biosciences, Gaithersburg, MD).
4
Gene Expression Analysis in Tissues and Cells
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RNA was extracted from tissue or cells using the RNeasy mini kit (Qiagen, Venlo, The Netherlands). In short, lysis was performed with Qiazol Lysis reagent (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich). RNA concentration and quality were determined with a Nanodrop spectrophotometer (Isogen Life Science, The Netherlands). cDNA synthesis was conducted using the Quanta qScript cDNA synthesis kit (Quanta Biosciences, Boston, MA, USA) per manufacturer’s instructions. qPCR was performed on a StepOnePlus™ Real-Time PCR system (Applied biosystems, Ghent, Belgium) using the SYBR green method (Applied Biosystems). The master mix contained 1× SYBR green, 10 µM primers, 12.5 ng cDNA, and nuclease free water. Data were normalized to the most stable reference genes and the ΔΔCt method was used to determine relative quantification of gene expression. Primers were designed using NCBI’s Primer-blast (details are shown in Table 1 ).
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