cDNA synthesis was performed as described in Balcells et al. [32 (link)], with the following reagents: 10ng of total RNA, Poly-A polymerase (NEB: M0276L), MulV RT enzyme (NEB M0253L) and dNTPs (NEB N0447L). The cDNA was diluted 10-fold for the RT-qPCR. The qPCR reaction was performed in RealQ Plus 2x Master Mix Green (without Rox, Ampliqon, A323406), with cycling conditions: 1x 95°C for 15 minutes, 40 cycles of (95°C for 30sec and 60°C for 30sec) using a Light Cycler 480 system (Roche).
Realq plus 2x master mix green without rox
Manufactured by Ampliqon
Sourced in Denmark
RealQ Plus 2x Master Mix Green without ROX is a ready-to-use master mix designed for real-time quantitative PCR (qPCR) experiments. It contains all the necessary components, including a hot-start DNA polymerase, dNTPs, and a green fluorescent dye for detection, optimized for reliable and sensitive qPCR results.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 system, by Roche (1 mentions)
Poly a polymerase, by New England Biolabs (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Cfx maestro software, by Bio-Rad (1 mentions)
Cfx connect real time pcr system, by Bio-Rad (1 mentions)
Rotor gene, by Qiagen (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
High pure rna tissue kit, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Revertaid first strand cdna synthesis, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 instrument system, by Roche (1 mentions)
Nucleospin rna plus mini kit, by Macherey-Nagel (1 mentions)
Quantstudio 5 machine, by Thermo Fisher Scientific (1 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Bead mill, by Qiagen (1 mentions)
6 protocols using realq plus 2x master mix green without rox
1
cDNA Synthesis and RT-qPCR Protocol
2
Quantitative RT-PCR for Viral RNA Detection
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The qRT-PCR was performed in the CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, United States) using a 25 μL reaction mix, including 12.5 μL of RealQ Plus 2x Master Mix Green without ROX (Ampliqon, Odense, Denmark), 0.75 μL each of primers (10 μM), 1 μL of cDNA, and 10 μL of RNase-free ddH2O. The reaction was carried out under the following conditions: one cycle at 95°C for 15 min for activation of the TEMPase hot-start enzyme, followed by 40 cycles of 95°C for 15 s, 61°C for 45 s, and 72°C for 20 s. The Ct values were calculated by the CFX Maestro Software (Bio-Rad, Hercules, CA, United States). The samples were subjected to melting curve analysis by heating the samples from 65 to 95°C following the final cycle of the PCR to detect specific and non-specific PCR products. Each sample was examined using three technical duplicates, and all plates contained virus-infected positive (with Kantar-05 and Yakutiye), healthy plant RNA, and no template samples as controls. The five-fold cDNA dilution series was used to determine the primer efficiency and standard curves. The qRT-PCR experiment was performed according to the conditions described above.
3
Quantification of SMG1 Expression in Patients
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Real-time PCR was used to evaluate SMG1 expression level in patients and
healthy individuals, using 7.5 µl of RealQ Plus 2x Master Mix Green Without ROX™
(Ampliqon, Denmark), 0.5 µl of each primer (forward and reverse), and 1 μl of cDNA, which
was adjusted using ddH2 O. Real-time PCR stages were conducted using ABI
Applied Biosystems™ (Thermofisher, USA) as bellow: 15 minutes at 95˚C for
pre-denaturation, and 19 seconds at 95˚C for denaturation, 19 seconds at 61.5˚C for
denaturation and extension, per cycle. The Rotor-Gene device (Qiagen, USA) was used to
perform thermal processes. Also, the GAPDH gene was used as the internal
control gene. The sequences of forward and reverse primers of SMG1 and
GAPDH genes are given inTable 1 .
healthy individuals, using 7.5 µl of RealQ Plus 2x Master Mix Green Without ROX™
(Ampliqon, Denmark), 0.5 µl of each primer (forward and reverse), and 1 μl of cDNA, which
was adjusted using ddH2 O. Real-time PCR stages were conducted using ABI
Applied Biosystems™ (Thermofisher, USA) as bellow: 15 minutes at 95˚C for
pre-denaturation, and 19 seconds at 95˚C for denaturation, 19 seconds at 61.5˚C for
denaturation and extension, per cycle. The Rotor-Gene device (Qiagen, USA) was used to
perform thermal processes. Also, the GAPDH gene was used as the internal
control gene. The sequences of forward and reverse primers of SMG1 and
GAPDH genes are given in
4
Quantitative RT-PCR Analysis of Gene Expression
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Cells were grown to 70 % confluence on 6-well plates and subjected to total RNA isolation using High Pure RNA Tissue Kit (Roche, Germany) following the manufacturer’s protocol.
Synthesis of cDNA with reverse transcriptase was performed by RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc).
For Real-Time quantitative RT-PCR reactions using the Real Q Plus 2x Master Mix Green, without ROX (AMPLIQON, Denmark) was applied on Rotor-Gene 6000 HRM Real Time
PCR Thermocycler (Corbett Life Science, Australia). Primers for candidate genes, used for qRT-PCR, were designated by Oligo 7 (version 7.56) software.
Details of the primers were listed inTable 1 Calculation of relative mRNA expression was performed by the 2-∆∆Ct method. All the mRNA expression values were
normalized to ß -actin as a reference gene.
Synthesis of cDNA with reverse transcriptase was performed by RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc).
For Real-Time quantitative RT-PCR reactions using the Real Q Plus 2x Master Mix Green, without ROX (AMPLIQON, Denmark) was applied on Rotor-Gene 6000 HRM Real Time
PCR Thermocycler (Corbett Life Science, Australia). Primers for candidate genes, used for qRT-PCR, were designated by Oligo 7 (version 7.56) software.
Details of the primers were listed in
normalized to ß -actin as a reference gene.
5
Quantifying Gene Expression Changes
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Total RNA was extracted from the treated cells with 5-ALA (250 µg/mL) for 7 days by the RNeasy Mini Kit (Qiagen, Hilden, Germany). Then, the RNA was reverse-transcribed using RevertAid First Strand cDNA Synthesis (Thermo Fisher Scientific, Wesel, Germany) and qRT-PCR with specific primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Bax, Bcl-2, p53, and cyclin D1 (Table 1 ) was accomplished using RealQ Plus 2X MasterMix Green-without Rox™ (Ampliqon, Odense, Denmark). A LightCycler® 96 Instrument system (Roche life Science, Indianapolis, IN, USA) was used for gene amplification, under the following conditions: 95 °C for 15 min, 40 cycles of 95 °C for 30 s, and annealing/extension at 60 °C for 60 s. The melting curve was generated at 60–95 °C for 6 s. GADPH was applied to normalize alterations in gene expressions. Using the 2−ΔΔCq technique, the fold change in gene expression was assessed [53 (link)].
6
RNA Extraction and qPCR Analysis
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Six replicate RNA preparations for each genotype or condition were prepared using the NucleoSpin RNA Plus Mini kit (Macherey-Nagel, #740984.50) kit; each sample contained 3-5 whole late feeding third-instar larvae or 5 larval brains. Animals or tissues were placed in 2-mL Eppendorf tubes containing lysis buffer + 1% betamercaptoethanol, and samples were lysed using a bead mill (Qiagen). RNA was purified according to the kit instructions, and cDNA was prepared using the High-Capacity cDNA Synthesis Kit (ThermoFisher, #4368814). QPCR reactions were prepared using RealQ Plus 2x Master Mix Green without ROX (Ampliqon, #A323402) and the gene-specific primers given in the STAR methods, and runs were performed using a QuantStudio 5 machine (Applied Biosystems).
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