Total RNA from OS tissues, the two OS cell lines MG-63 and U2OS and the human osteoblast hFOB 1.19 cell line was isolated with TRIzol® according to the manufacturer's instructions. Total RNA concentration was determined with photometric method using a NanoQuant plate (Tecan Group, Ltd.). RNA (1 ng) was reverse transcribed using the Reverse EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Trangene) according to the manufacturer's instructions. RT-qPCR was performed using SYBR-Green Master mix (Roche Life Science) in an ABI 7500 Real-time PCR instrument according to the following reactions: Denaturation for 1 cycle at 95°C for 1 min, followed by 40 cycles of 20 sec at 95°C and 40 sec at 58°C. GAPDH was used as an endogenous control. The primers used were designed as follows: AWPPH forward, 5′-CTGGATGGTCGCTGCTTTTTA-3′ and reverse, 5′-AGGGGGATGAGTCGTGATTT-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The relative expression levels were normalized to endogenous controls and assessed using the 2−ΔΔCq method (31 (link)).
Nanoquant plate
Manufactured by Tecan
Sourced in Switzerland, United States, Germany, Austria
The NanoQuant Plate is a lab equipment product designed for the quantification of nucleic acids and proteins. It provides a rapid and efficient method for measuring sample concentrations using small sample volumes. The NanoQuant Plate utilizes spectrophotometric technology to determine the concentration of samples.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Infinite m200 pro, by Tecan (8 mentions)
Infinite 200 m microplate reader, by Tecan (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Infinite m200 pro plate reader, by Tecan (5 mentions)
Infinite 200 pro, by Tecan (4 mentions)
Infinite m200 plate reader, by Tecan (4 mentions)
Infinite 200 pro plate reader, by Tecan (4 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (4 mentions)
Aligner software, by CodonCode (3 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (3 mentions)
X tremegene hp dna transfection reagent, by Roche (3 mentions)
Shodex kw 402.5 4f, by Resonac (2 mentions)
Infinity reader m200 pro, by Tecan (2 mentions)
Anykd mini protean tgx gels, by Bio-Rad (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Qiaamp powerfecal dna kit, by Qiagen (2 mentions)
Viia 7 system, by Thermo Fisher Scientific (2 mentions)
96 protocols using nanoquant plate
1
Quantifying AWPPH Expression in Osteosarcoma
2
Comprehensive DNA Extraction from Diverse Tissue Samples
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A pathologist determined benign and malign regions on HE-stained slides from fresh frozen tissue. Macrodissection was performed on consecutive slides (10μm slides with an average area of 10–15 cm2 of cancer and of benign tissue, respectively, which corresponded to ~5μg of DNA each). Then DNA was extracted from 85 tissue samples belonging to 28 patients with an EZ1 advanced Workstation with the EZ1 DNA tissue kit (QIAGEN, Hilden, Germany) and quantified with on a Tecan Infinity M200 with a Nano Quant Plate (Tecan Group Ltd. Männedorf, Switzerland). Tissue samples were taken from the primary tumors, the resection margins, the recurrences, the resection edges of the recurrences, blood, tumor areas in lymph nodes, benign areas in lymph nodes, from one second primary tumor and from one dysplasia (S1 Table
3
Genomic DNA Extraction and Bulk Sequencing
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Frozen leaf material from the F2 plants of the 24 selected F2:3 families was ground using a TissueLyser II (QIAGEN, Hilden, Germany) and DNA was extracted using the E.Z.N.A. HP Plant DNA Mini Kit (Omega Bio-Tek Inc., Norcross, GA). DNA was quantified with an Infinite M200 Pro plate reader (Tecan Group Ltd., Mannedorf, Switzerland) using the i-control software (Tecan Group Ltd.,) and a NanoQuant Plate™ (Tecan Group Ltd.,). Agarose gel electrophoresis was used to confirm the quality of genomic DNA. Equal amounts of DNA from the 12 most resistant and 12 most susceptible families were pooled to create the resistant (R-Bulk) and susceptible bulk (S-Bulk), respectively. The samples were sent to Novogene (Novogene Corporation Inc., Davis, CA) for whole genome sequencing on an Illumina Platform (PE150, Q30 > 80%).
4
Mechanical Stress Influences Gene Expression
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The impact of the mechanical treatment on gene expression was evaluated as follows: following the 24-h mechanical stretch, treated SAOS-2 cells and the static control counterpart were trypsin-detached, pelleted, processed, and -analyzed for the following target genes: COL1A1, RUNX-2, BGLAP/OCC, and ALPL. RNA extraction from cellular pellets was prepared using the TRIZOL Reagent (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s protocol. RNA quality was examined by measuring the absorbance ratio at 260 nm and 280 nm through the NanoQuant Plate of an Infinite®200 PRO multi-well plate reader (Tecan Group Ltd., Männedorf, Switzerland). RNA was reverse-transcribed with SensiFAST™ cDNA Synthesis Kit (Bioline, Meridian Bioscience, London, UK) following the manufacturer’s specifications. Gene expression was measured using iTaq Universal SYBR Green Supermix (Biorad Laboratories, Hercules, CA, USA). qRT-PCR was performed using a LightCycler 96 Real-Time PCR System (Roche Diagnostics GmbH). For data analysis, the expressions of all genes were normalized using the ΔΔ cycle threshold method against human glyceraldehyde 3-phosphate dehydrogenase GAPDH gene expression. The sequences of primers used are as follows.
5
Genomic DNA Extraction and Quantification
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Total DNA was isolated from the sample using the Pure Link genomic DNA extraction kit (ThermoFisher Scientific, USA) and stored at −80°C. Quantitative DNA measurements were performed using the Qubit dsDNA HS (high sensitivity) kit and Qubit 3.0 fluorimeter (both ThermoFisher Scientific, USA), according to the standard manufacturer's instructions. Since only the dsDNA/dsRNA‐bound form of the dye possesses intense fluorescence, there is no interference caused by other species in the solution. The A260/A280 ratio was measured on the Infinite 200 Pro device (Tecan Group Ltd., Switzerland) equipped with the NanoQuant plate (Tecan Group Ltd., Switzerland).
6
Characterization of MATCH Chain Dimerization
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Example 3
Efficient MATCH chain dimerization was further demonstrated by the remarkable homogeneity of the protein content in protein L-purified samples. The protein was analyzed over the course of four weeks and storage at 4° C. and 37° C. with respect to oligomerization by SE-HPLC and degradation by SDS-PAGE (see
7
Stability Assessment of MATCH Protein
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Example 3
Efficient MATCH chain dimerization was further demonstrated by the remarkable homogeneity of the protein content in protein L-purified samples. The protein was analyzed over the course of four weeks and storage at 4° C. and 37° C. with respect to oligomerization by SE-HPLC and degradation by SDS-PAGE (see
8
Comprehensive Gene Expression Analysis
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Total RNA isolation, cDNA synthesis and quantitative RT-PCR (qPCR) were performed according to established procedures48 (link). The primers were designed as described in the Supplementary Information (Table S2) and were synthesised by TIB Molbiol (Berlin, Germany). Fold differences in gene expression were normalised to their respective housekeeping gene, either YWHAZ, GAPDH, or RPLP0. Each housekeeping gene was selected based on its constant expression in all samples for each gene analysis. For PCR array analysis, the epidermis and dermis were separated or the NHDFs were trypsinized, and mRNA was extracted using a NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany). mRNA was quantified using a NanoQuant Plate and an Infinite200 PRO microplate reader (both Tecan Group Ltd., Männedorf, Switzerland). cDNA synthesis was performed using an RT2 First strand kit (Qiagen, Venlo, Netherlands). Age-related genes were studied using the Human Ageing RT2 Profiler PCR Array (Qiagen), and qPCR was performed using a StepOnePlus real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with the RT2 SYSB Green ROX qPCR MasterMix (Qiagen).
9
Genetic Analysis of CASP3 SNP
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Two milliliters of peripheral blood was taken from cases and controls for genetic analysis. Deoxyribonucleic acid (DNA) extraction was done using QIAmp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). DNA purity was checked using TECAN infinite M200 pro with Nanoquant plate (TECAN group, AG, Switzerland) taking the optical density of 260 nm and 280 nm. Samples were then stored at −800C.
Genotyping for the SNP rs113420705 was done using polymerase chain reaction (PCR) and Sanger sequencing. PCR amplification reactions were carried out at controlled conditions as per the standard protocol with the help of an automated PCR thermal cycle (Applied Biosystems, Thermo Fisher Scientific, Massachusetts, USA). PCR cycle conditions are available on request.
PCR products were then subject to purification and direct sequencing using the ABI Big Dye Terminator kit and ABI 3500 Gene Analyzer (Applied Biosystems, CA, USA). Sequencing results were analyzed using Codon Code Aligner software (Codon Code Corporation, Centerville, (MA). Primer sequences used for the amplification of the CASP3 gene comprising the SNP of interest are illustrated inTable 1 . Representative pictures of PCR and sequencing are depicted in Figure 1 .
Genotyping for the SNP rs113420705 was done using polymerase chain reaction (PCR) and Sanger sequencing. PCR amplification reactions were carried out at controlled conditions as per the standard protocol with the help of an automated PCR thermal cycle (Applied Biosystems, Thermo Fisher Scientific, Massachusetts, USA). PCR cycle conditions are available on request.
PCR products were then subject to purification and direct sequencing using the ABI Big Dye Terminator kit and ABI 3500 Gene Analyzer (Applied Biosystems, CA, USA). Sequencing results were analyzed using Codon Code Aligner software (Codon Code Corporation, Centerville, (MA). Primer sequences used for the amplification of the CASP3 gene comprising the SNP of interest are illustrated in
10
RNA Extraction and cDNA Synthesis
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After 15 days of culture, total RNA was extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For 2D and 3D hydrogels, RNA was extracted with QIAzol using Tissue Lyser (Qiagen) for 1 min at medium frequency. RNA concentration and quality were assessed using NanoQuant plate (Tecan Group Ltd., Männedorf, Switzerland). cDNA was obtained using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).
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