For gene expression analysis, total RNA was isolated with Trizol Reagent (Invitrogen). Template cDNA synthesis was performed using the NZY M-MuLV First-Strand cDNA Synthesis Kit (Nzytech, Lisbon, Portugal). Quantitative reverse transcription-PCR (qRT-PCR) was performed with the KAPA SYBR Fast Universal Kit from Sigma-Aldrich. (Madrid, Spain) All primers were purchased from Sigma-Aldrich and are described in Table S1 .
Nzy m mulv first strand cdna synthesis kit
Manufactured by NZYTech
Sourced in Portugal
The NZY M-MuLV First-Strand cDNA Synthesis kit is a laboratory tool used for the reverse transcription of RNA into complementary DNA (cDNA). The kit contains the necessary components, including the M-MuLV reverse transcriptase enzyme, to facilitate this process.
Automatically generated - may contain errors
Lab products found in correlation
Nzy total rna isolation kit, by NZYTech (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nzysupreme qpcr green master mix, by NZYTech (2 mentions)
Nzyol, by NZYTech (2 mentions)
Corbett rotor gene thermal cycler, by Qiagen (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Kapa sybr fast universal kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
1.4 mm ceramic beads, by Qiagen (1 mentions)
Magna lyser instrument, by Roche (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Trisure, by Meridian Bioscience (1 mentions)
Hot firepol evagreen qpcr mix, by Solis BioDyne (1 mentions)
Dna engine thermal cycler, by Bio-Rad (1 mentions)
13 protocols using nzy m mulv first strand cdna synthesis kit
1
Gene Expression Analysis Protocol
2
Quantitative RT-PCR Analysis of Gene Expression
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RNA extraction was carried out using NZY Total RNA Isolation Kit (Nzytech, Caparica, Portugal) following the instructions of the manufacturer. We used 100 ng of RNA for retrotranscription using NZY M-MuLV First-Strand cDNA synthesis kit (Nzytech, Caparica, Portugal). Real-time qPCR was carried out using PowerUp SYBR Green Master Mix (ThermoFisher, Waltham, USA), 1 μl of 1:10 cDNA dilution, and the primers shown in Supplementary Table S3 . The reaction was performed using a total volume of 15 μl in a Step-One Plus Real-Time PCR system (ThermoFisher, Waltham, USA), cycling conditions were as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 s, and 30 s at 60°C; followed by a melting curve. To normalize the expression of the KLF4, VDR, PPARγ, and EDN1, we followed the–ΔCT method using YWHAZ and ALAS1 as reference genes.
3
Lumbosacral DRG Neuron RNA Extraction
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Lumbosacral DRG neurons (L5-S1) were homogenized using a MagNa Lyser Instrument (Roche Diagnostics, Mannheim, Germany) and 1.4 mm ceramic beads (Qiagen, Hilden, Germany)). Total RNA was extracted from L5-S1 DRG tissue using the RNAqueous®-Micro kit (Applied Biosystems, Bedford, MA, USA). An aliquot of each RNA sample was run on a denaturing agarose gel to assess integrity. Intact RNA was then quantified by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), followed by treatment with DNase. DNase-treated RNA (500 ng) was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis Kit (NZYtech, Lisbon, Portugal), according to the manufacturer’s instructions. cDNA was diluted and stored at −20 °C for later use.
4
Anti-inflammatory Activity of Fucopol HMs
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The analysis of the anti-inflammatory activity of the Fucopol HMs in THP1 cells proceeded as previously described with a few modifications [66 (link)]. Briefly, 1 × 106 THP1 cells were seeded in 6-well plates, incubated for 2 h with 7 µg/mL Lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO, USA) and were then incubated for 30 min or 3 h with the Fucopol HMs (13 mm diameter) or with 500 µg/mL of Fucopol. Identical samples without the addition of LPS were prepared (-LPS) for negative control purposes. In another approach, cells were simultaneously exposed for 2 h to both LPS and the Fucopol HMs or to 500 µg/mL of Fucopol. After the incubation time, cells were pelleted via centrifugation (500× g, 5 min) and solubilized in 300 µL of NZYol (NZYtech, Lisboa, Portugal), and RNA was extracted according to the manufacturer’s instructions and reverse-transcribed with the NZY M-MulV First strand cDNA synthesis kit (NZYtech). The relative expression of the tumor necrosis factor α gene (TNF-α) and of the housekeeping gene RNA18S were determined using the Ct method (2−∆∆Ct) [67 (link)] after real-time quantitative amplification using the NZYSupreme qPCR Green Master Mix (NZYtech) in a Corbett Rotor-Gene thermal cycler (Qiagen, Hilden, Germany).
5
RNA Extraction and qPCR Analysis
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Total RNA was extracted with the NZY Total RNA Isolation kit (Nzytech), according to manufacturer’s instructions, quantified in a NanoDrop spectrophotometer (Thermo Fischer Scientific), and treated with RQ1 RNase-free DNase (Promega) for 30 min at 37°C, in accordance with manufacturer instructions. All primers, except TaqMan probes, were synthesized by Thermo Fisher. DNase I-treated RNA was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis kit (Nzytech) and Oligo(dT)20 primers. cDNA was amplified by real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystems) or NZYSupreme qPCR Green Master Mix (2x), ROX plus (Nzytech), according to manufacturer’s instructions. For primer details, see Table S5 . PrimerBank sequences were obtained from https://pga.mgh.harvard.edu/primeRbank/ .
6
Quantitative Analysis of Gene Expression
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Cells exposed to the different conditions (AuNP@PEG and Au-nanoconjugate) in 24-well plates were collected at different time points between 3 and 72 hr. Cells were centrifuged at 200 × g for 5 min at room temperature, and total RNA was extracted from cell pellets using TRIsure (Bioline) according to the manufacturer’s instructions. Total RNA (100 ng) was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis kit (NZYTech).
qPCR amplification of cDNA was performed on a Corbett Rotor-Gene 6000 thermal cycler (QIAGEN) using the HOT FIREpol EvaGreen qPCR Mix (Solis BioDyne) with the following primers, at a concentration of 100 nM each: BCR forward (5′-GAAGTGTTTCAGAAGCTTCTCC-3′) and ABL1 reverse (5′-GTTTGGGCTTCACACCATTCC-3′); BCL2 forward (5′-CTTCGCCGAGATGTCCAGCCA-3′) and BCL2 reverse (5′-CGCTCTCCACACACATGACCC-3′); 18S forward (5′-GTAACCCGTTGAACCCCATT-3′) and 18S reverse (5′-CCATCCAATCGGTAGTAGCG-3′). qPCR conditions included an initial denaturation at 95°C for 15 min and 40 cycles of 95°C for 20 s, 55°C (BCR-ABL1 and 18S) or 65°C (BCL-2) for 20 s, and 72°C for 30 s. qPCR data were analyzed by the Ct method (2−ΔΔCt), where relative gene expression is given by quantification of the gene of interest (BCR-ABL1 or BCL2) relative to internal control gene (18S), normalized to the control condition (cells exposed to AuNP@PEG).43 (link)
qPCR amplification of cDNA was performed on a Corbett Rotor-Gene 6000 thermal cycler (QIAGEN) using the HOT FIREpol EvaGreen qPCR Mix (Solis BioDyne) with the following primers, at a concentration of 100 nM each: BCR forward (5′-GAAGTGTTTCAGAAGCTTCTCC-3′) and ABL1 reverse (5′-GTTTGGGCTTCACACCATTCC-3′); BCL2 forward (5′-CTTCGCCGAGATGTCCAGCCA-3′) and BCL2 reverse (5′-CGCTCTCCACACACATGACCC-3′); 18S forward (5′-GTAACCCGTTGAACCCCATT-3′) and 18S reverse (5′-CCATCCAATCGGTAGTAGCG-3′). qPCR conditions included an initial denaturation at 95°C for 15 min and 40 cycles of 95°C for 20 s, 55°C (BCR-ABL1 and 18S) or 65°C (BCL-2) for 20 s, and 72°C for 30 s. qPCR data were analyzed by the Ct method (2−ΔΔCt), where relative gene expression is given by quantification of the gene of interest (BCR-ABL1 or BCL2) relative to internal control gene (18S), normalized to the control condition (cells exposed to AuNP@PEG).43 (link)
7
cDNA Synthesis from Total RNA
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The cDNA was synthesized from 100 ng of RNA using an NZY M-MuLV First-Strand cDNA Synthesis Kit (NZYtech, Lisbon, Portugal) for a final volume of 10 µL. Samples were incubated in DNA Engine® Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) at 25 °C for 10 min, 37 °C for 50 min, and 85 °C for 5 min. Afterwards, 0.5 µL of RNase H (NZYtech, Lisbon, Portugal) was added and samples were stirred in a vortex and incubated in a heat block at 37 °C for 20 min. The synthetized cDNA was stored at −20 °C for further analysis.
8
Gene Expression Analysis of Breast Tumors
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Cells or mouse tumors total RNA was extracted using the NZY Total RNA Isolation kit (#MB13402, Nzytech). RNA from human breast tumors was obtained from Biobanco-iMM. DNase I-treated RNA was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis kit (#MB17301, Nzytech) and Oligo (dT)20 primer; and cDNAs were amplified by real-time PCR using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) or NZY qPCR Green, ROX (#MB22003, Nzytech). Specific primers included: TNFRSF11A (#Hs00921372_m1, Applied Biosystems), TNFSF11 (#Hs00243522_m1, Applied Biosystems), PGR (#Hs01556702_m1, Applied Biosystems), ESR1 (#Hs01046816_m1, Applied Biosystems), Twist (Fw: 5′-ccggagacctagatgtcattg-3′; Rv: 5′-ccacgccctgtttctttg-3′), Slug (Fw: 5′-ccaaactacagcgaactgga-3′; Rv: 5′-gtggtatgacaggcatggag-3′), Vimentin (Fw: 5′-gaaaacaccctgcaatctt-3′; Rv: 5′-cctggatttcctcttcgtg-3′), N-cadherin (Fw: 5′-tatcgaaggatgtgcatga-3′; Rv: 5′-caggctcactgctctcata-3′), Oct4 (Fw: 5′-ctgagggcgaagcaggagtc-3′; Rv: 5′-cttggcaaattgctcgagtt-3′) and GAPDH (#PPH00150F, SA Biosciences). Gene expression was normalized using the housekeeping gene GAPDH, and relative mRNA expression was calculated using the 2–ΔΔCt method.
9
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted from CAM using TRIsure™ Reagent method (Bioline, London, UK) according to the manufacturer’s instructions. The quality of RNA was assessed using the Nanodrop ND-1000 Spectrophotometer (ThermoScientific, Wilmington, DE). Total RNA extracted from samples was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis kit (Nzytech, Lisbon, PT) followed by qPCR amplification using KAPA SYBR® FAST kit (KAPAbiosystems, Cape Town, SA). The following conditions were used: initial denaturation at 95 °C for 5 min; 40 cycles of 95 °C for 30 s, Tm °C for 30 s, 72 °C for 1 min; and a final extension step at 72 °C for 10 min. The Tm temperature varied from set of primers, as described in Additional file 1 : Table S1.5.
10
Hypoxia and Inflammation Gene Expression
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Inflammation and hypoxia markers HIF1A, RELA, VEGFA, MMP2, CTSD, IL6, and TNFA genes expression was assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). First, total RNA was extracted from 10 spheroids of each condition using SV Total RNA Isolation System (Promega Corporation, United States). Then, complementary DNA (cDNA) synthesis was achieved using NZY M-MulV First-Strand cDNA Synthesis Kit (nzytech, Portugal), following the manufacturer’s recommendations. cDNA amplification was performed using Rotor-Gene (Qiagen, Germany), using NZYSupreme qPCR Green Master Mix (2x) (nzytech, Portugal). The primers sequences and qPCR cycling programs used to evaluate each gene expression are described in the (Supplementary Tables S1, S2 ).
The RT-qPCR was used as endogenous control. Relative levels of gene expression were quantified based on the 2−ΔΔCT method (Schmittgen and Livak, 2008 (link)), using the 18S ribosomal RNA (18S) gene as endogenous control (Schmittgen and Zakrajsek, 2000 (link)).
The RT-qPCR was used as endogenous control. Relative levels of gene expression were quantified based on the 2−ΔΔCT method (Schmittgen and Livak, 2008 (link)), using the 18S ribosomal RNA (18S) gene as endogenous control (Schmittgen and Zakrajsek, 2000 (link)).
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