For RT-qPCR, eight primers designed using the gene sequence responsible to encode the proteins identified through MALDI-TOF, the sequences of designed primers are presented in Table 1 . In addition to these primers, antioxidative enzymes, superoxide dismutase (Cyt-SOD), catalase (CAT), ascorbate peroxidase, (APX) and glutathione reductase (GR), gene specific primers reported previously were used to study the respective gene expression changes under progressive water-deficit condition [28 ], sequence information is given in S1 Table . TRI reagent (Ambion) in combination with RNAase-free DNAase treatment (Qiagen, USA) were used for total RNA extraction. According to the manufacturer instructions cDNA synthesis kit (Bio-Rad, USA), were used for first-strand cDNA synthesis, for this in 20.0 μL reaction volume, 1 μg of total RNA was used. As per manufacturer’s instructions IQ SYBR Green Supermix (Bio-Rad, USA) were used for expression analysis, using iQ5 thermo cycler (Bio-Rad, Hercules, CA, USA) with iQ5 Optical System Software version 2.0 (Bio-Rad, Hercules, CA, USA).
Iq5 optical system software version 2
Manufactured by Bio-Rad
Sourced in United States
The IQ5 optical system software version 2.0 is a laboratory equipment product that functions as a real-time PCR detection system. It is designed to analyze and quantify nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Iq sybr green supermix, by Bio-Rad (5 mentions)
Icycler iq5 thermal cycler, by Bio-Rad (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr premix ex taq 2, by Takara Bio (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Myiq single color real time pcr detection system, by Bio-Rad (3 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (3 mentions)
Iq5 multicolor rt pcr detection system, by Bio-Rad (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Myiq2 two color real time pcr detection system, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Iq5 real time pcr detection system, by Bio-Rad (2 mentions)
Rnx plus, by Sinaclon (2 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Icycler iq5 real time pcr detection system, by Bio-Rad (2 mentions)
Iscript one step rt pcr kit, by Bio-Rad (2 mentions)
Iq5 thermocycler, by Bio-Rad (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
37 protocols using iq5 optical system software version 2
1
Investigating Antioxidative Gene Expression
2
Osteogenesis-related Gene Expression Analysis
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The expression of osteogenesis-related genes was evaluated using
RT-PCR as previously described.22 (link) MG63
cells were seeded at 2 × 104 cells/disc and cultured
for 7 or 14 days. Total RNA was isolated using TRIzol reagent (Thermo
Fisher Scientific). One microgram of RNA from each sample was reverse
transcribed into complementary DNA using the PrimeScript RT reagent
kit (TaKaRa Bio, Otsu, Japan). The expression levels of osteogenesis-related
genes, including ALP, RUNX2, and OCN, were quantified using an iQ5 Multicolor RT-PCR Detection
System (Bio-Rad Laboratories Inc.) with SYBR Premix Ex Taq II (TaKaRa
Bio). Data analysis was carried out using the iQ5 Optical System Software
Version 2.0 (Bio-Rad Laboratories Inc.). The relative expression levels
for each gene of interest were normalized to those of the housekeeping
gene (GAPDH). The primers used are listed inTable 2 .
RT-PCR as previously described.22 (link) MG63
cells were seeded at 2 × 104 cells/disc and cultured
for 7 or 14 days. Total RNA was isolated using TRIzol reagent (Thermo
Fisher Scientific). One microgram of RNA from each sample was reverse
transcribed into complementary DNA using the PrimeScript RT reagent
kit (TaKaRa Bio, Otsu, Japan). The expression levels of osteogenesis-related
genes, including ALP, RUNX2, and OCN, were quantified using an iQ5 Multicolor RT-PCR Detection
System (Bio-Rad Laboratories Inc.) with SYBR Premix Ex Taq II (TaKaRa
Bio). Data analysis was carried out using the iQ5 Optical System Software
Version 2.0 (Bio-Rad Laboratories Inc.). The relative expression levels
for each gene of interest were normalized to those of the housekeeping
gene (GAPDH). The primers used are listed in
3
Real-Time qPCR Expression Analysis
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For expression analysis, total RNA was extracted from pools of 15 larvae with TRIzol reagent (Thermo Fisher Scientific, 15596026). Poly(A) mRNA was purified from 5 μg of total RNA with the Dynabeads “mRNA direct kit” (Thermo Fisher Scientific, 61011) and used for cDNA synthesis with M-MLV Reverse Transcriptase RNase H- (Solis BioDyne, 06-21-010000) according to the manufacturer’s protocol. PCRs were performed with the SYBR green (Bio-Rad Laboratories) method in a CFX96™ Real-Time PCR thermal cycler (Bio-Rad Laboratories). ribosomal protein L13a (rpl13a) and ribosomal protein, large, P0 (rplp0) were used as internal standards in each sample in order to standardize the results by eliminating variation in mRNA and cDNA quantity and quality. The annealing temperature for PCR ranges from 58 to 60 °C, depending on the primer set used. The cycling parameters were 95 °C for 10 min, followed by 45 cycles at 95 °C for 30 s and annealing-extension for 30 s. No amplification products were observed in negative controls and no primer-dimer formations in the control templates. The data obtained were analysed using the iQ5 optical system software version 2.0 (Bio-Rad) including GeneEx Macro iQ5 Conversion and Genex Macro iQ5 files. All analyses were performed in triplicate. Primer sequences are reported in Table S1 .
4
Chondrocyte Gene Expression Analysis
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Samples of total RNA from chondrocytes seeded in cell culture plates or from freshly aspirated bone marrow were isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was reverse-transcribed into cDNA using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). The cDNA samples were amplified with a Pfu PCR kit (Tiangen, Beijing, China), and the specific primers were displayed in Table 1 . All PCR products were resolved on a 2% agarose gel.
Real-time PCR was performed on cDNA samples by using the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). PCR reactions were carried out on MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA) under the following conditions: cDNA was preheated for 15 min at 95°C, denatured for 5 min at 95°C, followed by 45 cycles, consisting of 15 s at 95°C, 15 s at 60°C, and 30 s at 72°C. For each reaction a melting curve was generated to test primer dimer formation and nonspecific priming. The primers for real-time PCR are listed inTable 1 . Calculation of relative expression was performed with Bio-Rad iQ5 optical system software (version 2.0) using the double delta Ct method [14 (link)]. GAPDH was used for normalization.
Real-time PCR was performed on cDNA samples by using the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). PCR reactions were carried out on MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA) under the following conditions: cDNA was preheated for 15 min at 95°C, denatured for 5 min at 95°C, followed by 45 cycles, consisting of 15 s at 95°C, 15 s at 60°C, and 30 s at 72°C. For each reaction a melting curve was generated to test primer dimer formation and nonspecific priming. The primers for real-time PCR are listed in
5
Real-Time PCR Expression Analysis
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cDNA samples obtained from RT-PCR were used in the Real Time quantitative PCR to determine gene expression after treatment. PCR amplification was carried out in 15 µL reaction volume containing cDNA, 1× iQ SYBR Green Supermix, and specific sense and anti-sense primers. The exception was TNF-α, whose amplification was carried out in 50 µL reaction volume.
All primer pairs used for Real Time PCR were designed using the software Beacon Designer 8.10 and they were synthesized by Sigma Genosys Ltd. Primer sequences, concentrations, and ng of cDNA used are reported inTable 1 . Standard curves were prepared using a ‘calibrator’ cDNA for each target and reference gene. In order to verify the specificity of the amplification, a melt-curve analysis was performed, immediately after the amplification protocol. Non-specific products of PCR were found in any case. The relative quantification was made using the Pfaffl modification of the ΔΔCt equation [43 (link)], taking into account the efficiencies of individual genes. The results were normalized in vivo to β-actin and Gapdh, and in vitro to β-actin and ribosomal subunit 18S, used as reference genes. The data were analyzed using iQ5™ optical system software Version 2.0 (Bio-Rad Laboratories Hercules, CA, USA).
All primer pairs used for Real Time PCR were designed using the software Beacon Designer 8.10 and they were synthesized by Sigma Genosys Ltd. Primer sequences, concentrations, and ng of cDNA used are reported in
6
Standardized qPCR Analysis of Genetic Markers
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PCRs were performed with the SYBR green method in an iQ5 iCycler thermal cycler (Bio-Rad laboratories). Triplicate PCRs were carried out for each sample following exactly Maradonna et al.53 (link). In addition, in order to standardize the results by eliminating variation in mRNA and cDNA quantity and quality54 (link), β-actin (actβ)55 (link) and acidic ribosomal protein (arp)56 (link) were used as the reference genes. The choice of reference genes were due based of the fact that their mRNA levels did not vary among developmental stages and experimental treatments. Results demonstrated neither amplification product produced in negative controls and nor primer-dimer formations in the control templates. The data obtained were analyzed using the iQ5 optical system software version 2.0 (Bio-Rad laboratories). Modification of gene expression is reported with respect to the control sample. The primer sequences for actβ, rplp, mgll, hnf4a, fit2, npy, gcga and nucb2a were designed using Primer3 (210 v. 0.4.0) (Supplemental Table 4 ).
7
Quantifying Inflammatory Cytokine Expression
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RNA was isolated via TRIzol® Reagent according to manufacturer’s instructions. For reverse transcription RevertAid H Minus First Strand cDNA Synthesis Kit from Thermo Scientific, Lithuania (EU) was used. qPCR was performed using the iQ5 real-time PCR detection system (Biorad), the PerfeCTa® SYBR Green SuperMix for iQ (Quanta Biosciences, Gaithersburg, USA) and the following primers: forward-IL-8∶5′-AGC TCT GTG TGA AGG TGC AG-3′, revers-IL-8∶5′-CTC TGC ACC CAG TTT TCC TT-3′; forward-TNFα: 5′-GCC CAG GCA GTC AGA TCA T-3′, reverse-TNFα: 5′- GCT GGT TAT CTC TCA GCT CCA-3′ ; forward-RPLP0∶5′-GCA ATG TTG CCA GTG TCT G-3′, reverse-RPLP0∶5′-GCC TTG ACC TTT TCA GCA A-3′. Data obtained were analysed with Bio Rad iQ5 Optical System Software version 2.0. The reference gene ribosomal protein, large, P0 (RPLP0) served for standardization of the individual PCRs. All assays were performed in duplicate.
8
Quantifying c-Myc mRNA in MCF-7 Cells
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MCF-7 cells were seeded in a 35-mm dish 1 day before transfection. To carry out the treatment, the cells were washed and then incubated further with different samples including blank culture media, free siRNA, negative control, CNPS, pRNA dimers, CN, and CNPPs at a siRNA concentration of 100 nM for 4 hr. After that, the cells were washed again with cold PBS and incubated further for 48 hr (for mRNA assays). The transfected cells were then collected. Then, c-Myc mRNA was evaluated with real-time qPCR.
The method for the real-time qPCR assay has been reported in the literature,36 (link) and the mRNA was assayed in this study with minor modifications. In brief, the analysis was carried out on the IQ5 Real-Time PCR Detection System (Bio-Rad), and the relative gene expression was quantified by the 2−ΔΔCt method using the IQ5 Optical System Software version 2.0 (Bio-Rad). The primers for PCR amplification were as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-GGGTGTGAACCATGAGAAGT-3′; GAPDH reverse: 5′-GACTGTGGTCATGAGTCCT-3′; c-Myc forward: 5′-GGCTATTCTGCCCATTTGGGGAC-3′; and c-Myc reverse: 5′-GGCAGCAGCTCGAATTTCTTC-3′. Reaction parameters were: 95°C for 10 s, then 61°C for 30 s, 40 cycles. Specificity was verified by melt curve analysis and agarose gel electrophoresis.
The method for the real-time qPCR assay has been reported in the literature,36 (link) and the mRNA was assayed in this study with minor modifications. In brief, the analysis was carried out on the IQ5 Real-Time PCR Detection System (Bio-Rad), and the relative gene expression was quantified by the 2−ΔΔCt method using the IQ5 Optical System Software version 2.0 (Bio-Rad). The primers for PCR amplification were as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-GGGTGTGAACCATGAGAAGT-3′; GAPDH reverse: 5′-GACTGTGGTCATGAGTCCT-3′; c-Myc forward: 5′-GGCTATTCTGCCCATTTGGGGAC-3′; and c-Myc reverse: 5′-GGCAGCAGCTCGAATTTCTTC-3′. Reaction parameters were: 95°C for 10 s, then 61°C for 30 s, 40 cycles. Specificity was verified by melt curve analysis and agarose gel electrophoresis.
9
Quantitative Real-Time RT-PCR Assay
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Total cellular RNA was isolated from cells using Trizol (Gibco-BRL). RNA was eluted with RNase-free water, quantified at an absorbance at 260/280 nm, and used for reverse transcription reaction. The primer sequences are present in Table 1 . Real-time quantitative reverse transcription-PCR was performed in an optional 96-well plate with Bio-Rad iQ5 system (Bio-Rad, USA) and a commercial SYBR-Green master mix kit (Takara, Japan), according to the manufacturer’s instruction. Assembled plates were then covered and run using the following conditions: an initial denaturation step of 95°C for 10 min followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. Each sample was measured in triplicate. The resulting data were analyzed with Bio-Rad iQ5 Optical System Software version 2.0 and shown as the mean ± SD of three determinations for each sample.
10
Quantitative Detection of DENV RNA in Mosquito Midgut
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To determine the presence of DENV RNA in the midgut of mosquitoes following an infectious blood meal, 2μL of midgut homogenate was heated at 95°C for 5 mins and chilled on ice prior to performing a one-step quantitative RT-PCR (qRT-PCR) reaction without RNA extraction. Presence of viral RNA was quantified using Brilliant III Ultra-Fast SYBR qRT-PCR Master Mix (Aligent Technologies, CA) and with the primer pairs for DENV2 RNA as described previously [109 (link)]. Quantitative RT-PCR was performed using a iQ5 real-time PCR detection system using the following cycles: RT at 50°C, 20 min; 95°C, 5 min for RT inactivation and 40 cycles of 95°C, 5 s and 60°C, 1 min. The results were analyzed with the iQ5 optical system software version 2.0 (Bio-Rad, CA).
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