Fetal bovine serum (FBS, F6178), L-glutamine, penicillin/streptomycin antibiotic, RPMI-1640 medium (R8758), and trypsin-EDTA were purchased from Biowest, France. Agarose and DMSO were purchased from Sigma-Aldrich Chemical Co., USA. Antibodies used for the detection of caspase-9 (primary mouse anti-human caspase-9 monoclonal antibody), microtubule-associated protein light chain 3- (LC3-) II (primary rabbit LC3-II oligoclonal antibody), and β-actin (primary rabbit anti-human β-actin monoclonal antibody) were obtained from eBioscience (Austria), Invitrogen (USA), and Sigma-Aldrich (USA), respectively. The primer sequences for Bcl-2, Beclin 1, vascular endothelial growth factor (VEGF), and GADPH were supplied by R&D systems (Minneapolis, MN, USA). Thermo Scientific Gene JET RNA Purification Kit (UK), Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (UK), and Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix kit (UK) were used in quantitative real time PCR analysis. Caspase-3 activity was measured using the colorimetric assay kit (R&D systems, USA). DNA fragmentation was performed by QIAamp DNA Mini Kit (QIAGEN, USA) using a suitable DNA marker (Gibco, BRL, Life technologies, USA). Other reagents were of analytical grade or the highest quality available.
Maxima sybr green rox qpcr master mix kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania, Germany
The Maxima SYBR Green/ROX qPCR Master Mix kit is a ready-to-use solution for real-time quantitative PCR (qPCR) analysis. It contains all the necessary components, including SYBR Green I dye and ROX passive reference dye, for sensitive and reliable gene expression analysis.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (16 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Steponeplus, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Rotor gene 6000, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Biophotometer, by Eppendorf (2 mentions)
Rnase free dnase, by Qiagen (2 mentions)
Yta total rna purification mini kit, by Yekta Tajhiz Azma (2 mentions)
Taq hot start dna polymerase, by Thermo Fisher Scientific (2 mentions)
Agarose, by Merck Group (1 mentions)
Colorimetric assay kit, by R&D Systems (1 mentions)
Penicillin streptomycin antibiotic, by Biowest (1 mentions)
Trypsin edta, by Biowest (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
33 protocols using maxima sybr green rox qpcr master mix kit
1
Apoptosis and Autophagy Pathway Analysis
2
Salinity Stress Response in Camelina
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Sterilized camelina seeds were planted at a depth of 2 cm in pots containing peat moss and were kept under the conditions of 16 h of light and a temperature of 25 °C with irrigation every three days. Then, the five-week-old seedlings were treated with salt (200 mM of NaCl) via irrigation, which was repeated after 24 h. After the salt treatment, leaves were collected at different time points (after 6, 24, and 72 h). The total RNA samples were extracted using an RNX kit (Sinaclon, Iran) and the cDNA was synthesized using a reverse transcriptase kit (Roche, Germany), according to manufacturer protocols. In the present study, five members of the SULTR family were selected for real-time PCR analysis. The genes were selected based on the phylogeny analysis. In addition, actin-2 (Csa15g026420) was used as a reference gene to normalize the expression data. Specific primers were designed using the Primer3 online software (version 4.1.0) [72 (link)], based on the coding sequences of the selected SULTR genes (Table S3 ). The expression patterns of the SULTR genes were analyzed using a Maxima SYBR Green/ROX qPCR Master Mix kit (Thermo Fisher, France) and the ABI Step One, according to manufacturer protocols. The expression levels of each SULTR gene were calculated using the delta Ct method [73 (link)], using three biological replicates.
3
Quantitative RT-PCR for Gene Expression
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TRIzol reagent (Invitrogen) was used to isolate and purify total RNA according to the protocols of the manufacturer. Thereafter, cDNA was synthesized using the complementary DNA synthesis kit (Thermo Fisher Scientific). mRNA expression was analyzed with the Maxima SYBR Green/ROX qPCR Master Mix kit (Thermo Fisher Scientific) on an Applied Biosystems (Thermo Fisher Scientific) Prism 7300 detection system, with GAPDH as the endogenous reference. The primer sequences: MKRN1(rat) 5′-ACTGTGGCCGTACTGCCCCTT-3′ (forward), 5′-GCATAGGGGCAAAGCTGCTTC-3′ (reverse); p21(rat) 5′-CGCCGCCGTGATGACCTGGGA-3′ (forward), 5′-CACGTGGTCCTCCGGAGCTGG-3′ (reverse); GAPDH(rat) 5′-GGGAAGCTCACTGGCATGGC-3′ (forward), 5′-GCCGCCTGCTTCACCACCTT-3′ (reverse); MKRN1(human) 5′-GAAGCACCCCTGCAGGGCTCA-3′ (forward), 5′-CTGCAGCATAGGGGCACAGCT-3′ (reverse); p21(human) 5′-GTGATGCGCTAATGGCGGGCT-3′ (forward), 5′-GTCACCCTCCAGTGGTGTCTC-3′ (reverse); GAPDH(human) 5′-ACAACTTTGGTATCGTGGAAGG-3′ (forward), 5′-GCCATCACGCCACAGTTTC-3′ (reverse); the relative gene expression level was calculated using the 2 − ΔΔCT method.
4
Comparative Gene Expression Analysis
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The relative expression of the genes was investigated using Real time PCR (Rotor gene Q-Pure Detection-Qiagen) in the treated plants compared with the control. Gene specific primer pairs were selected from previous studies [25 (link), 31 (link)]. Real time PCR reactions were carried out by considering three biological replications in the final volume of 12.5 μL using Maxima ® SYBR-Green/ROX qPCR Master mix kit (Thermo Fisher Scientific, USA), according to the manufacturer's instructions. Initial activation of the enzyme was done at 95°C for 10 min in one cycle, followed by 40 cycles including denaturation at 95°C for 10 s, annealing at 58–60°C for 15 s and fluorescence data collection at 72°C for 20 s. The actin gene was used as the reference gene to normalize the data. The relative expression of the studied genes was calculated after obtaining Ct by ΔΔCt method [74 (link)].
5
RT-qPCR Protocol for Gene Expression
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Total RNA was extracted from cell culture by utilizing Thermo Scientific Gene JET RNA Purification Kit, following the manufacturer's protocol. cDNA was generated with M-MuLV reverse transcriptase using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. For real time PCR quantification, Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix kit was used. Briefly, in a 25 μL reaction volume, 12.5 μL of master mix, 2.5 μL of primer assay, and 10 μL of template cDNA (100 ng) were added to each well. Sequences of primers were described in Table 1 . The PCR plate was subjected to 40 cycles of the following conditions: PCR activation at 95°C for 5 min, denaturation at 95°C for 5 sec, and annealing/extension at 60°C for 10 sec.
The values of RT-PCR products were normalized with respect to GAPDH and then compared to controls. The relative expression was calculated from the 2−ΔΔCT formula [21 (link)].
The values of RT-PCR products were normalized with respect to GAPDH and then compared to controls. The relative expression was calculated from the 2−ΔΔCT formula [21 (link)].
6
Total RNA Extraction and miRNA/mRNA Analysis
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Total RNA were extracted from all of the tissue fractions using miRCURY RNA Isolation Kit (Exiqon, Woburn, MA) or Purelink Mini RNA Kit (Invitrogen) according to the manufacturer protocol for purifying RNA from animal tissues. First-strand cDNA synthesis and real-time PCR for the total RNA were performed using Maxima First-strand cDNA synthesis and Maxima SYBR Green/ROX qPCR Master Mix kit (Thermo Scientific), respectively.
cDNA synthesis and real-time PCR for miRNA were performed using NCode miRNA First-strand cDNA synthesis (#MIRC-50, Sigma) and NCode SYBR GreenER qPCR SuperMix Universal (MIRQER-100). mRNA expression levels were calculated using ΔCt methods.
cDNA synthesis and real-time PCR for miRNA were performed using NCode miRNA First-strand cDNA synthesis (#MIRC-50, Sigma) and NCode SYBR GreenER qPCR SuperMix Universal (MIRQER-100). mRNA expression levels were calculated using ΔCt methods.
7
Validation of Microarray Data by qPCR
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Validation of microarray data was performed by qPCR, which was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the same cDNA products obtained as previously described (Section 2.9 ). The amplification was carried out with the primers listed in Supplementary Table S1 , while the reaction was performed using the Maxima SYBR Green/ROX qPCR Master Mix Kit (Thermo Fisher Scientific, Waltham, MA, USA) in 25 µL of total volume. The thermocycling was performed using a two-step cycling protocol: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s and 60 °C for 60 s. The last two steps were repeated for 40 cycles. All primers were designed using Primer 3 software. Two different reference genes actin1 (ACT1, At2g37620) and the elongation factor 1B alpha-subunit 2 (eEF1Balpha2, At5g19510) were used to normalize the results of qPCR; the most stable gene was ACT1. All amplification plots were analyzed with the QuantStudio Design and Analysis software (Applied Biosystem, Foster City, CA, USA) to obtain Ct values. Relative RNA levels were calibrated and normalized with the level of ACT1 mRNA.
8
Investigating Anti-Cancer Potential of nsLTP1 in MCF-7 and AsPC-1 Cells
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MCF-7 and AsPC-1 cells were seeded in 6-well plates at a density of 2.5 × 105 cells/well for 24 h. The cells were treated with different concentrations of nsLTP1 (5, 10, and 20 μM) for 48 h. TRIzol reagent (Invitrogen, USA) was used to extract mRNA according to the manufacturer’s protocol. The RNA concentration was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The yields were approximately 2 μg from each cell line which were reverse transcribed to cDNA using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific). The cDNA was used to quantify Bcl2, BAX, caspase-3, surviving, EGFR, and VEGF gene expressions by Real-Time Polymerase Chain Reaction (RT-PCR). Reactions were performed using Maxima SYBR Green/ROX qPCR master mix kit (Thermo Fisher Scientific) in QuantStudio 3 Real-Time thermocycler (Applied Biosystems, Thermo Fisher Scientific) under the following conditions: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 55 °C for 60 s. GAPDH was used as the housekeeping gene. Fold change in gene expression was calculated using the 2-ΔΔCt method and presented as the mean ± SD relative to control cells in three independent triplicate experiments [38 (link)].
9
Quantitative PCR Gene Expression Analysis
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qPCR was performed in Stratagene Mx3000P (Agilent Technologies, USA) instrument by using Maxima SYBR Green/ROX qPCR master mix kit (cat# K0221; Thermo Scientific) as per manufacturer’s protocol. After amplification, melting curve analysis was performed by following the method; 95 °C for 1 min, 55 °C and 95 °C for 30 s each. GAPDH was used as a reference gene for the normalization of data. Fold change in gene expression was calculated with the CT values obtained by ∆∆ct method.
10
Breast Cancer Cell Line Analysis by qRT-PCR
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The breast cancer cell lines MDA-MB-231 and BT-474 were seeded onto 100 mm Petri dishes at 60–70% confluence. Subsequently, the medium was replaced with hBM-MSC-cm. After 48 h, the total RNA was extracted using TRIzol-chloroform and treated with DNAse I (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized from 1 µg of total RNA using an oligo (dT18), following the supplier’s instructions (RevertAid First Strand cDNA Synthesis Kit, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of various genes were analyzed by qRT-PCR using the Maxima SYBR Green/ROX qPCR Master Mix Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control. The amplification conditions were as follows: denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Finally, the relative expression was calculated using the 2−ΔΔCT method [33 (link)]. The oligonucleotides for each analyzed gene were designed using the Oligo Analyzer Tool program and are shown in Table 2 .
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