Total RNA was extracted from lung cancer tissues using Trizol (Invitrogen, Inc). The amount and purity of RNA were measured spectrophotometrically on Ultrospec 5300 pro (Amersham Biosciences). Genomic DNA was removed from RNA samples by digestion with DNaseI, according to the manufacturer’s instructions. First strand cDNA was synthesized from 5 μg of total RNA using the oligo (dT) primers and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen, Inc) according to the manufacturer’s instructions. Real-Time PCR was used to quantify transcript levels and performed on the ABI StepOne real-time PCR using Absolute Blue SYBR green PCR mix (Thermo Fisher Scientific). The following primers were used: USP33 (5′-TGTGATGCTTAGGCAAGGAG-3′ and 5′-GGCCCTCCACCATAAATAGA-3′); Robo1 (5′-GCATCGCTGGAAGTAGCCATACT-3′ and 5′-CTAGAAATGGTGGGCTCAGGAT-3′); GAPDH (5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTGTTGTCATACTTCTCATGG-3′). We used a 2-step amplification (40 cycles of 95°C, 15 s; 60°C, 30 s; 72°C, 30 s; followed by melting temperature determination stage) and fold enrichment was calculated by the 2−ΔΔCT method.
Ultrospec 5300 pro
Manufactured by Cytiva
Sourced in United States
The Ultrospec 5300 pro is a UV/Visible spectrophotometer designed for a wide range of applications in life science laboratories. It features a wavelength range of 190 to 900 nm and can perform accurate absorbance measurements. The instrument provides a simple and intuitive user interface.
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Lab products found in correlation
M mlv rt, by Thermo Fisher Scientific (1 mentions)
Abi stepone, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
P 2000 polarimeter, by Jasco (1 mentions)
Autox hcn triple res inverse probe, by Agilent Technologies (1 mentions)
Maxis impact hd lc q tof mass spectrometer, by Bruker (1 mentions)
Oxford 600 mhz spectrometer, by Agilent Technologies (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
4 protocols using ultrospec 5300 pro
1
Quantifying Gene Expression in Lung Cancer
2
Spectroscopic Characterization of Compounds
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Optical rotations were measured on a JASCO P-2000 polarimeter, circular dichroism spectrum on a JASCO J-815, and UV spectra on an Amersham Biosciences Ultrospec 5300-pro UV/Visible spectrophotometer. NMR spectra were recorded with benzene or dimethylsufoxide as internal standards (δC 128.06, δH 7.16 for C6H6 and δC 39.52, δH 2.50 for DMSO) on a Varian Oxford 600 MHz spectrometer equipped with a 5 mm AutoX HCN triple res inverse probe (600 and 150 MHz for 1H and 13C NMR, respectively), and on a Varian VNMRS (Varian NMR System) 500 MHz spectrometer equipped with a broadband Cold Probe (500 and 125 MHz for 1H and 13C NMR, respectively). LR-LCMS data were obtained using an Agilent 1200 series HPLC system equipped with a photo-diode array detector and a 6130 quadrupole mass spectrometer. HRESIMS was carried out using a Bruker Maxis Impact HD LC-q-TOF Mass Spectrometer equipped with an uHPLC system. HPLC purifications were carried out using Agilent 1100 or 1200 series HPLC systems (Agilent Technologies) equipped with a photo-diode array detector. All solvents were of HPLC quality.
3
Quantification of Total Phenolic Content
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The extract fractions from fresh leaves (2 g) were obtained by percolation (96 h)
with 50 mL of methanol. After solvent evaporation, the Folin-Ciocalteu method
was used to determine total phenolic content (TPC) [44 ]. The extracts were solubilized again in
methanol at a concentration of 20 mg/mL and then diluted 30 times in water
because of our samples reactivity. This dilution should be tested for each type
of plant sample in order to maintain an absorbance between 0.3 and 0.7. Aliquots
of 500 μL of the diluted sample received 250 μL of Folin-Ciocalteu (1 N) and
then 1,250 μL of Na2CO3 (75 g/L). After 30 min of
incubation in the dark, the absorbance was read at 760 nm using a UV-Vis
spectrophotometer (Ultrospec 5300 pro, Amersham Biosciences, Little Chalfont,
Reino Unido). The experimental calibration curve was prepared using gallic acid.
TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of
extract (mg/GAE g−1) [44 ].
with 50 mL of methanol. After solvent evaporation, the Folin-Ciocalteu method
was used to determine total phenolic content (TPC) [44 ]. The extracts were solubilized again in
methanol at a concentration of 20 mg/mL and then diluted 30 times in water
because of our samples reactivity. This dilution should be tested for each type
of plant sample in order to maintain an absorbance between 0.3 and 0.7. Aliquots
of 500 μL of the diluted sample received 250 μL of Folin-Ciocalteu (1 N) and
then 1,250 μL of Na2CO3 (75 g/L). After 30 min of
incubation in the dark, the absorbance was read at 760 nm using a UV-Vis
spectrophotometer (Ultrospec 5300 pro, Amersham Biosciences, Little Chalfont,
Reino Unido). The experimental calibration curve was prepared using gallic acid.
TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of
extract (mg/GAE g−1) [44 ].
4
Characterization of Biogenic Silver Nanoparticles
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The formation of the AgNPs was monitored using UV–Vis spectral analysis. The spectra of cell-free secretomes treated with 2 mM AgNO3 were measured at 300–600 nm using an ultraviolet–visible (UV–Vis) spectrophotometer (Ultrospec 5300 pro, Amersham Biosciences Corp., Piscataway, NJ, USA). The size, distribution, and morphology of the nanoparticles were further analyzed via scanning electron microscopy (SEM) (Tescan Vega 3, Brno - Kohoutovice, Czech Republic). Samples for SEM were prepared by drop-coating the AgNP solution onto an aluminum grid. Before transferring samples to the microscope, they were dried at 55 °C. The zeta potential values of AgNPs were measured using a Malvern Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK) and recalculated applying the Smoluchowski model. The AgNP particle size distribution was evaluated using the dynamic light scattering (DLS) method (Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK) equipped with a 4 mW He-Ne laser emitting at a wavelength of 633 nm). Measurements were performed at 25 °C and an angle of 173°. These parameters were determined using AgNPs at a concentration of 1 mg/mL in water.
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