Total RNA was extracted from stimulated RAW cells with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and reverse transcribed to cDNA fragments using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Germany). The mRNA was amplified and quantified using the SybrGreen detection system on a QuantStudio 5 Real-time PCR System (Thermo Fisher Scientific) using the SybrGreen detection system (Thermo Fisher Scientific). Relative mRNA expression was calculated according to the ΔCt method. Relative values were normalized to the housekeeping gene eukaryotic translation elongation factor-2 (EEF2; forward primer 5’- agg cct gtg taa tat agc tgc g -3′, reverse primer 5′-ctc tgt gta gtt tgt agc tct gtc t-3′). Inducible NOS (NOS2) gene expression was detected with the RT2 qPCR Primer Assay for Mouse Nos2, NM_001313921 (QIAGEN).
Sybr green detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The SYBR Green detection system is a fluorescent dye used in real-time PCR to detect and quantify DNA amplification. It binds to double-stranded DNA, emitting a fluorescent signal that is proportional to the amount of DNA present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Mircury rna isolation kit tissue, by Qiagen (3 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (2 mentions)
Rnaiso plus reagent, by Takara Bio (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Universal cdna synthesis kit, by Qiagen (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
7900ht instrument, by Thermo Fisher Scientific (1 mentions)
Primer express 2, by Thermo Fisher Scientific (1 mentions)
Sds 2, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna xs, by Takara Bio (1 mentions)
32 protocols using sybr green detection system
1
Quantitative Analysis of Macrophage iNOS Expression
2
Liver RNA Extraction and qRT-PCR
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Total RNA of liver samples was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcribed to cDNA according to the manufacturer's instructions (TaKaRa Biotechnology Co., Ltd., Dalian, China). The mRNA expression in liver tissue was quantified using the SYBR Green detection system in 7900HT instrument (Applied Biosystems, Forster, CA, USA). The primer sequences are listed in Table 2 .
3
RNA Isolation and Real-Time PCR in Melanoma
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Melanoma cells (5×105) were lyzed in 1.0 mL Trizol® (Invitrogen, Paisley, UK). Total RNA was isolated according to the manufacturer's protocol (Life Technologies, Paisley, UK) utilizing Phase Lock Gel™ extraction tubes (Eppendorf AG, Hamburg, Germany). cDNA was prepared using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Warrington, UK) according to the protocol of the manufacturer. Real-time PCR was performed on ABI PRISM 7900HT using SYBR Green detection system (Applied Biosystems, Warrington, UK) essentially as described [25] (link). Primers were designed using “Primer Express 2.0” software (Applied Biosystems). Primer sequences are depicted in Table S1c in File S1 . Housekeeping genes (HKG) included eukaryotic translation elongation factor 1α1 (EF1A), actin beta (ACTB), and ribosomal protein (RPLP0). Each PCR reaction was performed in triplicates. For relative quantification, data were analyzed by ΔΔCT and ΔCT methods using SDS 2.1 software (Applied Biosystems). Expression levels of target genes were normalized to HKG expression levels (mean level of all 3 HKG examined).
4
Cardiac Tissue RNA Extraction and Analysis
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Total RNA was extracted from cardiac tissues by using Trizol reagent (Pars Tous, Mashhad, Iran), and then, RNA was converted to cDNA based on the manufacturer's protocol (Easy cDNA Synthesis Kit; Pars Tous, Iran). Real-time PCR was performed using a thermocycler and SYBR green detection system (Applied Biosystems, USA). Primer sequences were: 18S: 5ʹ-CGAACGTCTGCCCTATCAACTT-3ʹ, 5ʹ-CTTGGATGTGGTAGCCGTTTCT-3ʹ;PGC-1α: 5ʹ-CGGGATGGCAACTTCAGTAAT-3ʹ, 5ʹ-AAGAGCAAGAAGGCGACACA-3ʹ;NRF1: 5ʹ-GGGGAACAGAACAGGAAACA-3ʹ, 5ʹ-CCGTAATGCACGGCTAAGTT-3ʹ;NRF2: 5ʹ-GGGGAACAGAACAGGAAACA-3ʹ, 5ʹ-CCGTAATGCACGGCTAAGTT-3ʹ; and TFAM: 5ʹ-GAAAGCACAAATCAAGAGGAG-3ʹ, 5ʹ CTGCTTTTCATCATGAGACAG-3ʹ. The mRNA expression levels were indicated with 2-ΔΔCT and normalized to 18s rRNA [25 (link)].
5
Quantitative Analysis of Gene Expression in Mast Cells
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Total RNA was isolated from mast cells by using RNAiso Plus reagent (Takara) and NucleoSpin RNAXS (Takara). The cDNA was synthesized using SuperScript VILO cDNA synthesis kit (Life Technologies), and semiquantitative PCR was then performed using TAKARA Ex Taq HS DNA polymerase (Takara). The level of HDC, MCP5, CPA, Axin2, and Tcf7 mRNA was quantified using the SYBR Green detection system (Applied Biosystems). Results were normalized by expression of the GAPDH transcripts. The sequences of the primers used in this study are listed in Tables 1 and 2 .
6
Hippocampal microRNA and mRNA Profiling
7
Quantitative PCR Analysis of OPN-SV Transcripts
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Total RNA was extracted from cell lines and tumor tissues using Trizol reagent (Life Technologies, GIBCO BRL). For cDNA preparation, 1 μg of total RNA was reverse transcribed using the RevertAid first-strand cDNA synthesis kit (Fermentas, Burlington, ON, Canada).
Each OPN-SV transcript region was amplified with specific oligonucleotide pairs (Supplementary Table S3 and Supplementary Figure S1A ). Quantitative PCR reactions were conducted using SYBR Green detection system (Applied Biosystems, Warrington WA1 4SR, UK). Conditions for OPN-SV amplification were 50°C for 2 minutes, 94°C for 5 minutes followed by 40 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. Relative gene expression was calculated using the Delta-Delta CT method. GAPDH gene was used as the constitutive control.
Each OPN-SV transcript region was amplified with specific oligonucleotide pairs (
8
Quantitative Gene Expression Analysis
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Total RNA from the heat shock and oxidative stress samples was extracted by Trizol-chloroform method followed by DNase treatment. After assessing RNA quality, 1 μg of total RNA from each sample was used for cDNA synthesis (Takara PrimeScript first strand cDNA synthesis kit). mRNA expression levels were quantified by qRT-PCR on an ABI Prism 7900 HT (Applied Biosystems) using the SYBR Green detection system (Applied Biosystems). The reactions were performed according to the following conditions: initial hold at 95 °C for 30 s, followed by 40 cycles of amplification at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. At least three technical replicates were prepared in each group of samples. β-actin/RPL11 or TBP was used as a reference control. The relative mRNA expression was calculated using the 2-ΔΔct as a mean of at least three technical replicates. Primers for the target genes used for qRT-PCR are listed in Supplementary Information (Table S2 ).
9
Quantification of FAM83H-AS1, miR-136-5p, and MTDH
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Total RNA was extracted using TRIzol reagent (Invitrogen), and reverse transcribed into cDNAs by using a Reverse Transcription Kit with the M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative RT-PCR was utilized to determine the levels of FAM83H-AS1, miR-136-5p and MTDH with the SYBR Green detection system and the 7500 Real Time PCR System (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 were used as internal controls for mRNA or miRNA, respectively. The relative expression was measured using 2-ΔΔCt method. All experiments were conducted in triplicates.
10
Hippocampal miRNA and mRNA Expression
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