Total RNA was extracted with TRIzol according to the manufactureŕs instructions and quantified using Qubit reagent (Invitrogen). RNA was treated with DNase I (Ambion, Austin TX, USA) to remove potential contamination and reverse transcribed using random primers and the ImProm-II kit (Promega) to synthesize double-stranded cDNA. qPCR was performed with the commercial reagent Maxima SYBR Green qPCR Master Mix (Promega, Madison WI, USA) to determine GREM1, cyclophilin and GAPDH mRNA expression levels using the following primers: human GREM1 F (5′-CCCGGGGAGGAGGTGCTGGAGT-3′ ); human GREM1 R (5′-CCGGATGTGCCTGGGGATGTAGAA-3′ ); mouse cyclophilin1 F (5′-GCAGACATGGTCAACCCCACCG-3′ ); mouse cyclophilin1 R (5′-GAAATTAGAGTTGTCCACAGTCGG-3′ ); mouse GAPDH F(5′-TCCGCCCCTTCTGCCGATG-3′ ); and mouse GAPDH R (5′-CACGGAAGGCCATGGCAGTGA-3′ ). PCR product specificity was verified by melting curve analysis, and all of the real-time PCR reactions were performed in triplicate. The 2−ΔΔCT method was used to analyze the relative changes in gene expression levels [21] (link).
Qubit reagent
Manufactured by Thermo Fisher Scientific
The Qubit® reagents are a set of fluorescent dyes designed for quantifying nucleic acids and proteins in biological samples. The reagents bind to the target molecules, and the resulting fluorescent signal is proportional to the amount of the target present. The Qubit® system provides a sensitive and accurate method for determining the concentration of DNA, RNA, or proteins in a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Impromii kit, by Promega (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dntp mix, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Bravo liquid handler, by Agilent Technologies (1 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Mag bind total pure ngs beads, by Omega Bio-Tek (1 mentions)
Tape station reagents, by Agilent Technologies (1 mentions)
Sybr green 1 nucleic acid stain, by Merck Group (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Quantifluor dsdna system, by Promega (1 mentions)
Ethanol, by Carl Roth (1 mentions)
5 protocols using qubit reagent
1
Quantitative Gene Expression Analysis
2
Protein Digestion and Peptide Purification
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Proteins were solubilized with 7 M urea/2 M thiourea/5 mM TEAB solution. The samples were quantified by Qubit Reagent (Qubit® Quantitation Kit – Invitrogen) and 80 µg of proteins were submitted to the digestion procedure. First, proteins were reduced with 10 mM DTT for 1 h at 30 °C and alkylated with 40 mM IAA, in the dark, for 30 min at room temperature. After this, the mixture was diluted 10× with 50 mM TEAB. The trypsin was added with a proportion of 1:50 w/w and the reaction was carried out for 18 h at 35 °C. The reaction was stopped with formic acid with a final concentration of 1%. Lastly, sample peptides were desalted using C18 Reverse Phase Chromatography Micro SpinColumns (Harvard Apparatus).
3
RNA Extraction and Library Prep
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RNA extraction was performed as previously outlined (Mossman, Tross et al. 2016 (link)). Briefly, we extracted RNA from 30, 5-day-old healthy flies in each genotype x hypoxia × sex treatment. There were three replicates per genotype × hypoxia × sex condition, with the exception of one sample of siI;AutW132 females, which had two replicates at hypoxia timepoint = 0; resulting in a total of 71 RNA libraries. RNA extraction followed a modified RNA-Seq sample preparation protocol from the Gilad Laboratory (Marioni et al. 2008 (link)). Messenger RNA was first extracted, followed by RNA fragmentation, cDNA first strand synthesis, second strand synthesis, end repair, poly adenylation, adapter ligation and PCR enrichment. Throughout, RNA and DNA were quantified using the Qubit® Kits (RNA Broad Range, ds DNA Broad Range, and ds DNA High Sensitivity) with a Qubit® 1.0 Fluorometer. All Qubit® reagents were obtained from Molecular Probes™ (ThermoFisher Scientific). Following PCR enrichment, we size selected PCR products with size range = 334–500 bp using a Caliper Lab Chip XT (DNA 750 chip) (Caliper Life Sciences, Inc. Hopkington, MA, USA).
4
Comprehensive DNA Library Preparation
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DNA libraries were purchased from TriLink Biotechnologies and all DNA primers were purchased from Integrated DNA Technologies with HPLC purification. All peptides were purchased from Genscript. 10X PBS and Tween-20 were purchased from Sigma-Aldrich. Lambda Exonuclease and buffer were purchased from New England Biolabs. Mag-Bind Total Pure NGS beads were purchased from Omega-Biotek. The bioanalyzer and all reagents, the Bravo liquid handler, and Herculase II Phusion polymerase and buffer were purchased from Agilent. Tubes, plates, and thermocyclers were purchased from Eppendorf. Nunc plates were purchased from VWR. Both 70% and 200 proof ethanol was purchased from Fisher Scientific. Nuclease-free water, MgCl2, Bovine Serum Albumin, dNTP mix, Dynabeads M280 Streptavidin, and QuBit reagents were purchased from Thermo Scientific.
5
Plant Genomic DNA Extraction and Sequencing
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Gibberellic acid 4 + 7 (Duchefa Biochemie)
Ethanol (Carl Roth)
Paper bags for bagging plants (HERA)
Bird protection mesh (mesh size 25 mm) (Zill GmbH Co. KG)
Insect protection mesh (mesh size 0.6 mm) (Grow it)
2 ml and 1.5 ml tubes
96 well PCR plates (Sarstedt)
Foil/lids to seal plates
384 Well Lightcycler plates (Sarstedt)
Adhesive Optical film (Biozym Scientific)
Nuclease-free water
Liquid nitrogen
Quiagen DNeasy Plant Mini Kit (Quiagen)
AMPure XP beads (Beckmann Coulter)
Magnetic stand (Applied Biosystems)
KAPA HiFi Hotstart PCR Kit with dNTPs (Roche)
DMSO (Carl Roth)
ROX solution (ThermoFisher Scientific)
SYBR Green I Nucleic acid stain (Sigma-Aldrich)
TE buffer (10 mM Tris–HCl, pH 7.5)
QuantiFluor dsDNA System (Promega)
Tapestation reagents (Agilent Technologies)
Qubit reagents (ThermoFisher Scientific)
NextSeq Reagent Kit (Illumina)
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