qRT-PCR was run with MESA BLUE qPCR Master Mix Plus for SYBR® Assay No ROX (Eurogentec, Seraing, Belgium) on LightCycler 480 Real-Time PCR System (Roche Molecular Diagnostics, Pleasanton, CA, USA). The relative telomere length, T/S ratio, is the ratio of the telomere repeat copy number (T) to the copy number of a single copy gene (S) [38 (link)].
Mesa blue qpcr master mix plus for sybr assay no rox
Manufactured by Eurogentec
Sourced in Belgium, Germany, United States
MESA BLUE qPCR Master Mix Plus for SYBR® Assay No ROX is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments using the SYBR® Green detection method. It contains all the necessary components, including a DNA polymerase, buffer, and SYBR® Green I dye, for efficient and reliable qPCR amplification without the need for a passive reference dye.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Lunascript rt supermix kit, by New England Biolabs (1 mentions)
Qscript cdna supermix, by Quanta Biosciences (1 mentions)
Cfx384 real time system, by Bio-Rad (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 rt pcr system, by Roche (1 mentions)
4 protocols using mesa blue qpcr master mix plus for sybr assay no rox
1
Telomere Length Quantification by qRT-PCR
2
RNA Extraction and qPCR Analysis
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RNA was extracted from 4 leaf disks according to manufacturer instructions using the GeneMATRIX Universal RNA Purification Kit (Roboklon) with on‐column DNase I digestion.
RNA integrity was checked by loading on 1% agarose gel and separating by electrophoresis. RNA concentrations were measured using Nanodrop 2000 (Thermo Fisher), and equal amounts of RNA were used for cDNA synthesis. cDNA synthesis was performed using LunaScript™ RT SuperMix Kit (New England Biolabs) and in a standard thermocycler according to manufacturer instructions. Gene expression was measured by qPCR using MESA BLUE qPCR MasterMix Plus for SYBR® Assay No ROX (Eurogentec) and cycle quantification by Biorad CFX system.
RNA integrity was checked by loading on 1% agarose gel and separating by electrophoresis. RNA concentrations were measured using Nanodrop 2000 (Thermo Fisher), and equal amounts of RNA were used for cDNA synthesis. cDNA synthesis was performed using LunaScript™ RT SuperMix Kit (New England Biolabs) and in a standard thermocycler according to manufacturer instructions. Gene expression was measured by qPCR using MESA BLUE qPCR MasterMix Plus for SYBR® Assay No ROX (Eurogentec) and cycle quantification by Biorad CFX system.
3
qRT-PCR for Validating RNA-Seq Gene Expression
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To confirm gene expression levels detected by RNA-Seq, quantitative real-time PCR was performed in a Bio-Rad CFX 384TM Real-Time System (Bio-Rad, Munich, Germany) using gene-specific oligonucleotides (Additional file 9 ). The cDNA for qRT-PCR analyses was synthesized from 1 μg total RNA with the qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) using the same RNA samples as for the cDNA library construction. Each PCR reaction contained 4 μl MESA Blue qPCR™ Mastermix Plus for SYBR Assay no ROX (Eurogentec, Cologne, Germany), 1 μl cDNA sample and 100 nM gene-specific oligonucleotide primers to a final volume of 8 μl. The primer efficiency of each oligonucleotide was calculated using the following dilution series: 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and 1/128. The relative expression levels of the transcripts were calculated with reference to the housekeeping gene myosin (Genbank AC: 486090G09.x1). Significant differences in gene expression levels were determined by a two-sided Student’s t-test.
4
Gene Expression Analysis of HAPE and HL
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Gene expression was performed on 40 samples each of HAPE-p, HAPE-f, and HLs. Total RNA was extracted from an aliquot of 2 mL whole blood by TRI reagent RT blood (Molecular Research Centre, Cincinnati, OH, USA). RNA quantity and quality were determined on a NanoDrop ND-1000 spectrophotometer, and integrity was checked by running on 1.5% agarose gel. Total RNA, 5.0 μg, was used to generate cDNA by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) for real-time polymerase chain reaction (qRT-PCR) of several genes. The primers for qRT-PCR were designed using the NCBI primer BLAST browser. The primers and optimal conditions for amplification are presented in Supplementary Table S2 . qRT-PCR was performed in triplicate for each gene and each sample on LightCycler 480 RT-PCR System (Roche Molecular Diagnostics, Pleasanton, CA, USA) using MESA BLUE qPCR Master Mix Plus for SYBR® Assay No ROX (Eurogentec, Seraing, Belgium). Relative transcript quantity was calculated using the ΔΔCt method with 18SrRNA as an endogenous reference gene.
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