The total RNA of non-infected DH82 cells and DH82 CDV-Ond pi, DH82 CDV-Ondneon pi, DH82 CDV-Ondneon-vasostatin pi and DH82 CDV-Ondneon-GM-CSF pi cells was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and purified with RNeasy Mini Kit (Qiagen, Hilden, Germany) and RNase free DNase Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. The RNA concentration was spectroscopically measured at 260 nm using the GeneQuant pro (GE Healthcare, Amersham, Buckinghamshire, United Kingdom). Transcription to cDNA was performed using Omniscript (Qiagen, Hilden, Germany), RNAse OUT (ThermoFischer Scientific, Schwerte, Germany) and Random Hexamers (Promega, Madison, WI, USA) according to the manufacturer’s protocols. Reverse transcription was performed using a Biometra Thermocycler T-Gradient ThermoBlock (American Laboratory Trading, East Lyme, CT, USA) under the following conditions: 25 °C for 10 min, 37 °C for 1 h and 93 °C for 5 min.
Genequant pro
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The GeneQuant pro is a UV/Visible spectrophotometer designed for quantification and purity analysis of nucleic acid samples. It measures the absorbance of samples at multiple wavelengths to determine the concentration and purity of DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Random hexamer, by Promega (2 mentions)
Rnaseout, by Thermo Fisher Scientific (2 mentions)
Omniscript, by Qiagen (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Rna isolation reagent, by Takara Bio (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase kit, by Qiagen (1 mentions)
M mlv reverse transcriptase omniscript, by Qiagen (1 mentions)
Nucleospin rna 2, by Macherey-Nagel (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 0, by Merck Group (1 mentions)
18 protocols using genequant pro
1
RNA Extraction and cDNA Synthesis
2
RNA Isolation and Quantitative PCR Analysis
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Total RNA was isolated from a tissue according to the manufacturer’s protocol (NucleoSpin RNA II; Machery-Nagel, Bethlehem, PA, USA). Concentration and purity were assessed by spectrophotometric analysis (Genequant-Pro, GE Healthcare, Milan, Italy). One μg of total RNA was reverse-transcribed to cDNA with M-MLV Reverse Transcriptase (Omniscript; QIAGEN, Milan, Italy) using random primers in a 20 μL reaction incubated at 25 °C for 5 min followed by 60 min at 42 °C and 5 min at 70 °C. Two hundred ng of cDNA was amplified as follows: 15 min at 95 °C, 1 min of denaturation at 94 °C, 30 s of annealing, and 30 s of extension at 72 °C for 38 cycles. Primer sequences are summarized in Table 1 . Amplification products were highlighted with ethidium bromide on 1.5% agarose gel. The intensities of the bands were quantified by densitometric analysis. The used histamine receptor gene sequences are reported in Table 1 .
3
RNA Extraction and cDNA Synthesis
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Total RNA was extracted according to the protocol of TRIzol reagent (Invitrogen Corporation, Beijing, China) after ensuring that the tumour tissue contained more than 75% of tumour cells and had been approved by pathologists in the pathology department. RNA quality and concentration were measured by GeneQuant Pro (GE Healthcare, Buckinghamshire, United Kingdom). cDNA was synthesised from 1.0 μg of total RNA in a 20 μl reaction mixture using the PrimeScript RT Reagent Kit with gDNA eraser (Takara Bio Inc., Otsu, Japan).
4
RNA Extraction and cDNA Synthesis
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Total RNA was extracted according to the protocol of TRIzol reagent (Invitrogen, Beijing, China) after ensuring that tumors contained more than 75% tumor cells and approved by pathologists in the pathology department. RNA quality and concentration were measured using a GeneQuant pro (GE Healthcare, Piscataway, NJ, USA). cDNA was synthesized from 1.0 μg of total RNA in a 20 μL reaction mixture using a PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Otsu, Japan).
5
RNA Isolation and cDNA Transcription
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Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNA digestion with RNase free DNase (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. The RNA concentration was spectroscopically measured at 260 nm using the GeneQuant pro (GE Healthcare, Amersham, Buckinghamshire, UK). RNA to cDNA transcription was performed according to the manufacturer’s protocols with OmniScript (Qiagen, Hilden, Germany), RNAseOut (ThermoFischer Scientific, Schwerte, Germany), and Random Hexamers (Promega, Madison, WI, USA). Reverse transcription was performed using a Biometra Thermocycler T-Gradient ThermoBlock (American Laboratory Trading, East Lyme, CT, USA) under the following conditions: 25 °C for 10 min, 37 °C for 1 h, and 93 °C for 5 min.
6
Plasma miRNA Quantification by qRT-PCR
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Total RNA was extracted from 350 μL of plasma using TRIzol LS reagent (Invitrogen) and dissolved in 10 μL of DEPC (diethylprocarbonate) water. The quantity and quality of total RNA were determined with the use of a spectrophotometer (GeneQuant pro, GE Healthcare). miRNAs were quantified by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) TaqMan miRNA Assays (Applied Biosystems) according to the manufacturer’s instructions. Ten nomograms of total RNA were used to synthesize miRNA-specific complementary DNA (cDNA). PCR was performed in duplicates for 40 cycles using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The Ct value was defined as the cycle number at which the fluorescence (ΔRn) exceeded the threshold. The threshold was 0.20, which was defined as the default setting. To evaluate the miRNA expression levels, PCR was carried out in duplicate, and the average of the Ct values was converted into 2−Ct. If the Ct value could not be determined because the PCR was run for up to 40 cycles and the miRNA expression level was still below the detection limit, the Ct value was treated as 40 (i.e., miRNA expression level = 2–40).
7
Collecting Drosophila Larval Excretory Secretions
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Fly eggs were washed with ultrapure water, 1% sodium hypochlorite solution, 70% ethanol, and ultrapure water. The washed eggs were placed in a plastic tube. One hundred first instar larvae that hatched from eggs were incubated in 200 μl of PBS for 1 h at room temperature. The supernatant containing larval ES was collected in a 1.5 ml tube. The larval ES solution was concentrated using a centrifugal filter Amicon Ultra-0.5 (UFC500324, Merck Millipore, Darmstadt, Germany). The protein concentration of the larval ES solution was determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific, MA, USA) and a spectrometer GeneQuant pro (GE Healthcare, IL, US) in accordance with the manufacturers’ protocols. The larval ES solution was aliquoted and stored at − 30 °C until use.
8
Secretome Protein/Nucleic Acid Quantification
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The secretome aliquots were used to assess the protein/DNA/RNA content. Measurements were performed at 280/260-nm wavelength using NanoDrop™ (Thermo Scientific) which allows micro-volume (1 µl) sample measurements and at the same time by a spectrophotometer (Gene Quant pro, GE Healthcare. Quartz cell 5-mm pathlength). Each time, three assays were performed on the same sample set for comparison.
9
Quantification of OX1R and OX2R mRNA in SH-SY5Y Cells
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Total ribonucleic acid (RNA) was isolated from SH-SY5Y cells using RNeasy Mini kits according to the manufacturer's instructions. The purity and concentration of RNA were measured using a Gene Quant pro device (Nanovue Spectrophotometer, GE Healthcare, London, UK). Complementary deoxyribonucleic acid (cDNA) was synthesized with 2.5 μg total RNA using the SuperScript™ III First-Strand Synthesis System according to standard protocol. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a detector (Bio-Rad, Munich, Germany). The PCR solution contained specific primers (0.5 μl each/10 μmol/L), 4.0 μl 2.5 × SYBR Green Real Master Mix, and 0.25 μL cDNA with a final volume of 10 μL. The following primers were used: Human OX1R sense: 5’-CAG CCT ATG TGG CTG TGT TCG T-3’, antisense: 5’-AGT TGG TGA CTG TCC TCA TGT GG-3’); human OX2R sense: 5’-GTG TTC GTC GTG GCT CTC ATT G-3’, antisense: 5’-CAG GTG ATG GTC ACG AGC ACA T-3’) and human β-actin sense: 5’-CTG GAA CGG TGA AGG TGA CA-3’, antisense: 5’-AAG GGA CTT CCT GTA ACA ATG CA-3’. The reaction conditions for amplifying DNA were 94°C for 2 minutes, followed by 45 cycles of 94°C for 20 seconds, 62°C for 20 seconds, and 72°C for 30 seconds. The messenger RNA (mRNA) expression was normalized to the expression level of β-actin and was calculated using the following equation: Fold change = 2−ΔΔCT.
10
RNA and DNA Extraction from Liver Tissues
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The frozen samples were stored at −70°C until required for analysis. Total RNA and DNA
were extracted from female liver tissues using the RNeasy Mini kit (QIAGEN K.K., Tokyo,
Japan) and the DNeasy Tissue Kit (QIAGEN K.K.), respectively. The concentrations of RNA
and DNA were measured using a spectrophotometer (GeneQuant pro; GE Healthcare Japan,
Tokyo, Japan). These nucleic acid samples were stored at −70°C until required.
were extracted from female liver tissues using the RNeasy Mini kit (QIAGEN K.K., Tokyo,
Japan) and the DNeasy Tissue Kit (QIAGEN K.K.), respectively. The concentrations of RNA
and DNA were measured using a spectrophotometer (GeneQuant pro; GE Healthcare Japan,
Tokyo, Japan). These nucleic acid samples were stored at −70°C until required.
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