RNA was extracted, quantified and reverse transcribed as previously described using TRIzol® Plus RNA Purification System (Life Technologies, Paisley, UK) and iScript cDNA synthesis kit (Bio-Rad, Hemel-Hempstead, UK) after DNase treatment (Promega, Southampton, UK) following the manufacturers protocol (Mathew et al., 2016 (link)). cDNA (1 μg per sample) was amplified in triplicate using iTaq universal SYBR Green Supermix and CFX Connect Real-Time System (Bio-Rad, Hertfordshire, UK). Primers and reaction conditions are listed in Supplementary Table SI . Melt curves were obtained for all genes from 65°C to 95°C in 0.5°C increments for 5 seconds. Relative mRNA transcript expression for CA9, VEGFA and PR was calculated using the ΔΔCT method, relative to the reference genes, beta actin (ACTB) and peptidylprolyl Isomerase A (PPIA) and normalized to Ishikawa cell line expression using Bio-Rad CFX Manager (Bio-Rad, Hertfordshire, UK).
Dnase treatment
Manufactured by Promega
Sourced in United States, United Kingdom, Germany
DNase treatment is a laboratory procedure used to remove DNA from RNA samples. It is a crucial step in RNA purification, ensuring the removal of any contaminating DNA that may interfere with downstream applications, such as reverse transcription and real-time PCR.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (17 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
M mlv reverse transcriptase, by Promega (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Cfx96, by Bio-Rad (4 mentions)
Iq sybr green supermix, by Bio-Rad (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Sybr green mix, by Roche (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Cfx96tm real time system, by Bio-Rad (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Lightcycler 480, by Roche (3 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Bio-Rad (2 mentions)
Mrna seq sample preparation kit, by Illumina (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Isogen, by Nippon Gene (2 mentions)
One step sybr prime script tm rt pcr kit 2, by Takara Bio (2 mentions)
62 protocols using dnase treatment
1
Quantitative Gene Expression Analysis
2
RNA Extraction and qPCR Analysis
3
RNA Extraction and qPCR Analysis of 3T3-L1 Adipocytes
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Total RNA was extracted from mature, fully differentiated 3T3-L1 adipocytes on day 8 using the TRIzol (Invitrogen, Carlsbad, CA, USA) method [82 (link)]. DNA contamination was removed using the DNase treatment (Promega, Madison, WI, USA). Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) was used to determine the concentration and purity (260/280) of extracted RNA, and RNA integrity was confirmed using 1.2% agarose gel. The gene expression analysis was performed using the Bio-Rad CFX96TM Real-Time System. Primers used in the real-time quantitative polymerase chain reaction (qPCR) were designed using NCBI primer blast, and purchased from IDT Technologies (Coralville, IA, USA). The efficiency of all primers was within the acceptable range of 90–110%. Primer sequences are presented in Supplementary Table S1 . Amplification was carried out using iQ SYBR Green Supermix (# 1708880, Bio-Rad, Hercules, CA, USA), and a reaction volume was 10 μL with 50 ng of cDNA per reaction as per our previous publications [83 (link)]. Data analyses were carried out using the CFX Manager TM Software, version 3.0 (Bio-Rad, Hercules, CA, USA). The delta Ct values for each gene of interest were obtained, and the mRNA expression of target genes was normalized to RPLP0 as the reference gene, a large ribosomal protein. The expression of target genes was calculated using the ΔΔCt method [84 (link)].
4
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol (Thermo Fisher), and 0.5 μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega) after DNase treatment (Promega). Subsequently, qPCR was performed on the cDNA using SYBR Green Mix (Roche) on CFX96 (Bio-Rad) real-time PCR machine. The primers are listed in Supplementary Table S1 .
5
Porcine Muscle Transcriptome Profiling
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Total RNA was extracted from a 100 mg sample of LD muscle using TRIZOL reagent (Invitrogen, Paisley, UK) according to manufacturer instructions, followed by DNase treatment (Promega, Southampton, UK). Total RNA from all 164 pigs (5 time points, 3 treatment groups) was processed using Agilent Technologies one-color Low Input Quick Amp Labelling Kit according to the manufacturer protocol. Cyanine 3-CTP labelled cRNA was quantified using the NanoDrop 8000 (Thermo Scientific) and 1.65 μg of cRNA was then hybridized onto Agilent Technologies porcine gene expression (V2) 4 × 44 k microarrays at 65 °C for 17 hours in a SciGene Model 777 Microarray Oven. Arrays were processed in a SciGene NoZone Workspace, to limit exposure to ozone, and washed and dried using the Little DipperTM Microarray Processor, Model 650C. Arrays were scanned with the Agilent Technologies DNA Microarray C Scanner at a resolution of 5 μm.
6
Quantitative Gene Expression Analysis of hBMSCs
7
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, UK) according to the manufacturer’s protocol. Obtained RNA was subjected to DNase treatment (Promega, USA). Subsequently, the purified RNA was reverse transcribed with murine leukemia virus reverse transcriptase (Invitrogen, the Netherlands).
The mRNA expression was quantified by SYBR Green (BioRad, USA) real-time PCR on a CFX96 real-time detection system (BioRad, USA). Real-time PCR primers (Biolegio, the Netherlands) were designed with Primer 3 software (Whitehead Institute for Biomedical Research, USA). Primer sequences are provided inS1 Table . Obtained mRNA levels were normalized by glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as an endogenous control. Relative mRNA expression was analyzed according to the Livak method (2-ΔΔcT) and annotated as times-fold change of expression compared to control [31 (link)].
The mRNA expression was quantified by SYBR Green (BioRad, USA) real-time PCR on a CFX96 real-time detection system (BioRad, USA). Real-time PCR primers (Biolegio, the Netherlands) were designed with Primer 3 software (Whitehead Institute for Biomedical Research, USA). Primer sequences are provided in
8
RNA Extraction and cDNA Synthesis
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Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, California, USA) followed by DNAse treatment (Promega, Madison, Wisconsin, USA). The reverse polymerase transcription was performed using MMLV reverse transcriptase (Invitrogen, Carlsbad, California, USA) according to manufacturer's protocol.
9
Transcriptomic Analysis of Wheat-Stripe Rust Interaction
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The three Pt isolates (S96, S108, and S473) and wheat cultivar Chinese Spring (without the resistance genes Lr1, Lr15 and Lr24) were used for RNA sequencing. For each isolate, infected leaves were harvested at 3, 5, and 7 days after inoculation and immediately stored in liquid nitrogen. Samples were ground to a fine powder in liquid nitrogen and total RNA from rust pathogen and wheat leaf was extracted with the ISOLATE II RNA Mini Kit (Bioline, London, UK). After DNase treatment (Promega, Madison, WI, USA), RNA was purified by on-column DNase treatment and the quality was checked using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). About 10 μg of total RNA was processed with the Illumina mRNA-Seq Sample Preparation kit for library preparation. Each library was sequenced using the Illumina HiSeq 2500 platform (125 bp paired-end reads) at Ramaciotti Centre for Genomics (Sydney, Australia).
10
Quantitative Analysis of Gene Expression
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The total RNA isolation was performed using RNeasy Mini kit (Qiagen, Hilden, Germany). followed by DNAse treatment (Promega, Madison, WI, USA). RNA (1 μg) was used for the reverse transcriptase reaction that was carried out using M-MLV reverse transcriptase (Promega) according to the manufacturer’s protocol. qRT-PCR analysis was performed on ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA, USA) using TaqMan Gene Expression Master MiX (Applied Biosystems, Inc.). Probes used in this study were as follows: human GAPDH (Hs99999905_m1), MMP9 (Hs00234579_m1), TIMP1 (Hs00171558_m1), TIMP2 (Hs00234278_m1), HIF-1α (Hs00153153_m1), VEGF (Hs00900055_m1) and mouse GAPDH (Mm99999915_g1). The mRNA expression level was normalized to the housekeeping gene GAPDH. The experiments were performed three times in duplicate. Data are presented as % of control cells (wild-type).
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