The Caco-2BBe, C2PLKO.1 and C2NHE2KD cells were seeded at the density of 1.5 × 104/cm2 and 3 × 104/cm2, respectively, and cultured on transwell membranes (pore size 0.4 μm) for 14 days. On the 14th day, cells were harvested for RT-PCR analysis. Cell lysate was homogenized with QIAshredder homogenizer and the total RNA was extracted from the cells using the RNeasy® Mini Kit (Qiagen GmbH) following the guidance of the instruction manual. RNA quality was determined by capillary gel electrophoresis with the QIAxcel® system (Qiagen GmbH). 1 μg RNA was reverse transcribed with the QuantiTect® Reverse Transcription Kit (Qiagen GmbH) and cDNA concentration was measured spectrophotometrically. cDNA was diluted 1:20 with DNase free water and 2 μL of the dilution was used as a template for PCR. Each reaction additionally contained 5 μL 2× MESA Green qPCRMasterMix (Eurogentec GmbH) and an appropriate amount of primers (Supplementary Table 1 - for all supplemental Material see www.cellphysiolbiochem.com ).
Mesa green qpcr mastermix
Manufactured by Eurogentec
Sourced in Belgium
MESA GREEN qPCR MasterMix is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains all the necessary components for efficient and reliable qPCR, including a DNA polymerase, buffer, and fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect reverse transcription kit, by Qiagen (8 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Emt rt2 profiler pcr array, by Qiagen (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Qiaxcel system, by Qiagen (2 mentions)
Sds v2, by Thermo Fisher Scientific (2 mentions)
Abi 7900ht fast qpcr system, by Thermo Fisher Scientific (2 mentions)
Rq manager v1, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Matlab software, by MathWorks (1 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
17 protocols using mesa green qpcr mastermix
1
Quantifying Gene Expression in Cell Lines
2
RNA Isolation and qRT-PCR Analysis of MYB Genes
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Total RNA was isolated from the samples by using a Spectrum Plant Total RNA Kit (Sigma-Aldrich, Lt. Louis, MO, USA) according to manufacturer’s instructions. The RNA quality was checked on a 1.2% agarose gel with 18% formaldehyde. The purity and yield were estimated by a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA samples with a 260/280 nm ratio from 2.0 to 2.1 were used for further analysis. RNA purification was done by treating with DNAse I (Sigma-Aldrich, MO, USA) as per the manufacturer’s protocol.
A cDNA synthesis was carried out by a First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). An expression analysis of selected MYB genes was performed using the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All the primers used in this study were designed by the PrimerQuest tool of IDT. The qRT-PCR reaction mixture included a total volume of 20 μL containing 2 μL (20 ng) of cDNA, 10 μL of SYBR premix buffer (Mesa Green qPCR Master Mix (Eurogentec)), 1.0 μL each of forward and reverse primers (5 μM) and 6 μL of nuclease-free water. The qRT-PCR run profile was as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. EF1α (elongation factor 1 alpha) and GAPDH (glyceraldehyde 3-phosphatedehydrogenase) [51 (link)] genes were taken for data normalization.
A cDNA synthesis was carried out by a First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). An expression analysis of selected MYB genes was performed using the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All the primers used in this study were designed by the PrimerQuest tool of IDT. The qRT-PCR reaction mixture included a total volume of 20 μL containing 2 μL (20 ng) of cDNA, 10 μL of SYBR premix buffer (Mesa Green qPCR Master Mix (Eurogentec)), 1.0 μL each of forward and reverse primers (5 μM) and 6 μL of nuclease-free water. The qRT-PCR run profile was as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. EF1α (elongation factor 1 alpha) and GAPDH (glyceraldehyde 3-phosphatedehydrogenase) [51 (link)] genes were taken for data normalization.
3
RNA Extraction and qPCR Analysis of EMT Genes
4
Quantitative Real-Time RT-PCR Protocol
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Quantitative real-time RT PCR was performed on RNA samples using a two-step procedure. RNA was first reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer’s instructions. qPCR was then conducted on the cDNA in a 384-well plate with a ABI-7900HT Fast qPCR system (Applied Biosystems, Foster City, CA, USA). Mesa Green qPCR MasterMix (Eurogentec, Liège, Belgium) was added to the cDNA (5 μL for every 2 μL of cDNA). The following amplification conditions were used: 95 °C for 5 min; 40 cycles of 95 °C for 15 s, 57 °C for 20 s, and 72 °C for 20 s; 95 °C for 15 s; 60 °C for 15 s; and 95 °C for 15 s. Primer sequences for genes that were used in the study are given in Supplementary file 3 . The output Ct values and dissociation curves were analysed using SDS v2.3 and RQ Manager v1.2 (Applied Biosystems). Gene expression data were normalised against the 40 s ribosomal protein S17 (RS17) housekeeping gene, and compared with mock controls using the comparative CT method (also referred to as the 2−ΔΔCT method [27 (link)]). All samples were loaded in triplicate.
5
Q-PCR Quantification of Plant mRNA Transcripts
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In vitro grown plantlets were used in all Q-PCR experiments. Plants were gathered and RNA was extracted with the Qiagen RNeasy plant mini kit (Qiagen, cat 74903, Hilden, Germany). The “user-developed protocol” for plant tissue was used without modifications. 1 µg of total RNAs were reverse transcribed using the Superscript II kit (Invitrogen, 18064, Carlsbad, CA, USA), according to the manufacturer’s instructions. mRNAs were quantified by Q-PCR using the MESA GREEN qPCR MasterMix (Eurogentec, RT-SY2X-03+WOU, Liege, Belgium). Actin and 26S proteasome mRNA were both used as reference genes in all experiments. Runs were performed on the CFX384 Touch™ Real-Time PCR Detection System and relative mRNA levels were analysed using the software Bio-Rad CFX manager (http://www.bio-rad.com , accessed on February 2016). Primers used for Q-PCR are presented in Supplementary Table S2 .
6
Quantitative RT-PCR for Gene Expression
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Quantitative real-time RT PCR was performed on RNA samples using a two-step procedure. RNA was first reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer’s instructions. qPCR was then conducted on the cDNA in a 384-well plate with a ABI-7900HT Fast qPCR system (Applied Biosystems). Mesa Green qPCR MasterMix (Eurogentec) was added to the cDNA (5 μl for every 2 μl of cDNA). The following amplification conditions were used: 95 °C for 5 minutes; 40 cycles of 95 °C for 15 seconds, 57 °C for 20 seconds, and 72 °C for 20 seconds; 95 °C for 15 seconds; 60 °C for 15 seconds; and 95 °C for 15 seconds. Primer sequences for genes that were used in the study are given in Supplementary Table 4 . The output Ct values and dissociation curves were analysed using SDS v2.3 and RQ Manager v1.2 (Applied Biosystems). Gene expression data were normalized against the housekeeping gene GAPDH, and compared with the mock controls using the comparative CT method (also referred to as the 2−ΔΔCT method65 (link)). All samples were loaded in triplicate.
7
Gene Expression Analysis of BRD Proteins
8
Comprehensive RNA Expression Profiling
9
Axenic DNA Isolation and qPCR for Ustilago maydis
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For axenic cultures, U. maydis DNA isolation was performed as previously described (Tsukuda et al. 1988 (link)) from cell cultures at the indicated times. For qPCR, 1 µg of genomic DNA was used per reaction using specific primers and the SsoAdvanced Universal SYBR Green Supermix (BioRad) in a CFX96 Real-Time PCR system (BioRad). Reaction conditions were as follows: 3 min 95°C followed by 40 cycles of 10 s 95°C/10 s 60°C/30 s 72°C.
For qPCR assays of plant-infected fungal genomic DNA, infected plant tissue (100–300 mg) was harvested seven days post-inoculation, shock frozen with liquid nitrogen, and ground into a fine powder using a mortar. Genomic DNA was isolated from ground powder as described previously (Hoffman and Winston 1987 (link)). qPCR was performed using MESA GREEN qPCR MasterMix (Eurogentec) in a CFX96 Real-Time PCR system (BioRad). Cycling conditions were as follows: 7 min 95°C followed by 45 cycles of 30 s 95°C/20 s 60°C/40 s 72°C; specificity of PCR products was checked by melting curve analysis from 55°C to 95°C.
For qPCR assays of plant-infected fungal genomic DNA, infected plant tissue (100–300 mg) was harvested seven days post-inoculation, shock frozen with liquid nitrogen, and ground into a fine powder using a mortar. Genomic DNA was isolated from ground powder as described previously (Hoffman and Winston 1987 (link)). qPCR was performed using MESA GREEN qPCR MasterMix (Eurogentec) in a CFX96 Real-Time PCR system (BioRad). Cycling conditions were as follows: 7 min 95°C followed by 45 cycles of 30 s 95°C/20 s 60°C/40 s 72°C; specificity of PCR products was checked by melting curve analysis from 55°C to 95°C.
10
Quantitative Real-Time PCR Workflow
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Total RNA was extracted using TRIzol (Invitrogen) and treated with TURBO DNA-free (Ambion). cDNA was generated using qScript cDNA synthesis kit (Quanta Biosciences). qPCR was performed with Mesa Green qPCR master mix (Eurogentec) with specific primers listed in (primers Additional file 1 : Table S1). When possible, primers were designed to encompass exon-exon junctions. Cq values were normalized to GAPDH housekeeping reference gene and changes in expression level were calculated using the 2-∆∆CT method [29 (link)]. All data were obtained from 3 independent differentiations with PCR carried out in 2 independent runs each with three technical replicates. All PCRs were run on a QuantStudio Real-time PCR machine (Applied Biosystems).
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