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Genorm reference gene selection kit

Manufactured by Primerdesign
Sourced in United Kingdom

The GeNorm Reference Gene Selection Kit is a laboratory tool designed to identify the most stable reference genes for gene expression normalization in qPCR experiments. The kit provides a systematic approach to assess and select the optimal reference genes for a given experimental setup.

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6 protocols using genorm reference gene selection kit

1

Adipose and Liver RNA Extraction

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RNA was extracted from adipose tissue with TRI Reagent combined with the RNeasy Mini Kit (Qiagen Ltd.), and from liver using RNeasy Mini Kit following manufacturer's instructions. On-column DNase digestion was performed using RNase-Free DNase set (Qiagen Ltd.), and RNA concentration and purity assessed using a NanoDrop One spectrometer (ThermoFisher Scientific, UK). Complimentary DNA was synthesised using TaqMan Reverse Transcription Kit (Applied Biosystems, UK) as described previously (Hogg et al., 2012) (link). To select the most stable housekeeping genes the geNorm Reference Gene Selection Kit (Primerdesign Ltd., UK) was used, identifying the suitability of the geometric mean of ACTB and MDH1 for liver and SAT, and RPS26 and 18S for VAT.
Primers (Supplementary Table 1) were designed and synthesised as described previously (Siemienowicz et al., 2020) (link). Quantitative RT-PCR was performed on 384-well plate format (Applied Biosystems) with all samples assayed in duplicate and housekeeping control genes included in each run, as well as template, RNA and RT-negative controls, using the ABI 7900HT Fast Real Time PCR system (Applied Biosystems) as described previously (Hogg et al., 2012) (link). The transcript abundance of target gene relative to the housekeeping genes was quantified using the Ct method (Livak and Schmittgen, 2001) (link).
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2

Standardized qRT-PCR Gene Expression

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RNA extraction and quantitative (q) RT-PCR were performed as previously reported [27 ,32 (link)]. The input value of the gene of interest was standardized to a housekeeping gene calculated using GeNorm reference gene selection kit (PrimerDesign Ltd, Southampton, UK). (N = 3 experiments). The specific sequences of primers used in this study are included in S1 Table.
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3

Oxidative Stress Assay in BEAS-2B Cells

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Bronchial Epithelial Basal Medium and BEGM™ BulleKit™ were purchased from Lonza (Basel, Switzerland); Human bronchial epithelial cells BEAS-2B from ATCC (Manassas, VA, USA); tert-butyl hydroperoxide, Glutathione Fluoriometric Assay Kit, NaHCO3, HEPES, and non-essential amino acids were obtained from Sigma-Aldrich (St. Louis, MO, USA); WST-1 Proliferation Assay and High Fidelity cDNA synthesis Kit from Roche (Mannheim, Germany); 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate and Hank’s Balanced Salt Solution from Thermo Fisher Scientific (Waltham, MA, USA); NucleoSpin RNA II from Macherey-Nagel (Düren, Germany); Illumina Human-HT12 v4 Expression BeadChips were from Illumina (San Diego, CA, USA); Illumina TotalPrep RNA Amplification Kit from Ambion (Austin, TX, USA); RT-qPCR master mix, PerfectProbe assays and geNorm Reference Gene Selection Kit from Primerdesign (Southampton, UK); Dulbecco’s Modified Eagle’s Medium and Gentamicin Sulfate from Gibco (Paisley, UK) and Luciferase Assay kit from BioThema (Handen, Sweden).
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4

Real-time qPCR analysis of mRNA expression

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Analysis of mRNA expression was also conducted at the CMG, Antwerp University Hospital. Total RNA was isolated from whole blood (5 mL) collected in PAXgene tubes (PreAnalytiX, Hombrechtikon, Switzerland) with the aid of the PAXgene Blood RNA kit (PreAnalytiX) and the DNA-free DNA Removal Kit (Thermo Fisher). Total RNA quality was checked by electrophoresis using the Experion RNA StdSens kit (Bio-Rad, California, USA). Given the relatively low RNA yield, isolated RNA was additionally concentrated using the RNA Clean & Concentrator kit (Zymo Research). This total RNA was then converted to cDNA using the SuperScript III First-strand Synthesis System (Thermo Fisher) with 300 ng of RNA as starting material. Forward (GATGACAACTTGACTTCTCTGG) and reverse (AGCTTACATCTGGTCTCATGCT) primers were developed and tested, yielding a mean amplification efficiency of approximately 100% [E = 2.040, SE(E) = 0.022] and adequate Cp values (<30) for cDNA concentrations of 100 ng/µl. Next, reference genes were selected using the geNorm Reference Gene Selection Kit and software (PrimerDesign, Camberley, UK). From this experiment TOP-1, UBC and ACTB were selected as the most stably expressed genes in our sample. Consequently, a two-step real-time PCR experiment was performed with the aid of the qPCR MasterMix Plus for SYBR Green I No ROX (Eurogentec, Seraing, Belgium).
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5

Validating Microarray Assays with qPCR

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To validate the microarray assays, synthesis of cDNA was performed using 5 µg of total- RNA previously used for microarrays. cDNA was generated using Superscript III as described by the manufacturer (Invitrogen). Primer pairs were synthesised by PrimerDesign or Qiagen (QuantiTect Primer Assay), and the sequences of primers or the Qiagen ID are shown in Supplementary Tables 1A and 1B, respectively. The assays were performed using the ABI PRISM 7700 therocycler (ABI, USA), carrying out the method as described in the SYBR Green SuperMix-UDG Kit (Invitrogen). Primer efficiency was tested and a range between 90 and 110% was considered acceptable. The housekeeping gene for qPCR normalisation was selected using GeNorm reference gene selection kit (Primerdesign), and gene Gapdh was found the least variable housekeeping gene. Quantity calculations were performed using the REST (relative expression software tool) software (Pfaffl et al. 2002 ). Statistical calculation of probability of differential expression were based on a randomisation of samples using the Pair Wise Fixed Reallocation Randomisation Test (Pfaffl et al. 2002 ). REST was set for a number of 1000 randomisations during this analysis.
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6

Immunofluorescence Staining of Muscle Cells

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Dulbecco’s modified Eagle’s medium (DMEM) low glucose variation, Dulbecco’s phosphate buffered saline (PBS), trypsin–ethylenediaminetetraacetic acid (EDTA) preparation and foetal bovine serum (FBS) were obtained from PAA (Pasching, Austria). Adult horse serum, goat serum, paraformaldehyde, Triton X-100, bovine serum albumin and diamidinophenylindole (DAPI) were obtained from Sigma–Aldrich (Dorset, UK). Complete mesenchymal stem cell growth medium was obtained from Promocell (Heidelberg, Germany). ProLong Gold antifade reagent, AlexaFluor-488 conjugated goat-anti-mouse IgG antibody and AlexaFluor-546 goat-anti-rabbit IgG antibody were all purchased from Molecular Probes (Life Technologies, Paisley, UK). DNAse I and DNAse buffer were obtained from Ambion (Life Technologies). Monoclonal mouse-anti-rabbit fast skeletal myosin (myosin heavy chain (MHC)) antibody, monoclonal mouse-anti-mouse (synthetic fragment) MyoD1 antibody and monoclonal rabbit-anti-human lamin A (human only) primary antibody were obtained from Abcam (Cambridge, UK). MicroMACS one-step complementary DNA (cDNA) kit was purchased from Miltenyi Biotec (Surrey, UK). Precision 2X PCR Master Mix with SYBR Green, GeNorm reference gene selection kit and custom designed primers for quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were obtained from Primer Design Ltd (Southampton, UK).
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