Rox reference dye

Manufactured by Takara Bio

Sourced in Japan, China, United States

The ROX Reference Dye is a fluorescent dye used as a passive reference in quantitative real-time PCR (qPCR) experiments. It provides a stable fluorescent signal that is used to normalize the target gene signal, improving the accuracy and precision of qPCR data analysis.

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78 protocols using rox reference dye

Soil Microbiome Genomic Analysis

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Soil genomic DNA was extracted from 0.5 g of soil samples on days 0 and 28 with the RNeasy Powersoil DNA Elution kit (Qiagen, Germany). The extracted DNA concentration and purity were evaluated using Qubit4.0 Fuorometer (Invitrogen, United States).
The abundances of the key nitrifying including ammonia oxidizers and nitrite oxidizers were quantified using the ABI 7500 system (ABI, United States). Target genes, primers, and thermocycling conditions are shown in Supplementary Table 2. The total PCR reaction volume was 20 μL, which contains 10 μL 2 × SYBR Premix Ex Taq (Takara, China), 0.5 μL 50 × Rox Reference Dye (Takara, China), 0.4 μL forward and reverse primers, 2 μL template DNA (10–20 ng) and 7.2 μL RNase-free water. PCR products of targeted genes (AOA, AOB, Comammox amoA and Nitrobacter-like NOB nxrA and Nitrospira-like NOB nxrB) were inserted into PMD18-T plasmids. Standard curves were constructed using 10-fold serial dilutions of plasmids DNA from one representative clone containing the correct targeted gene. Melting curve analysis was conducted between 65 and 95°C at the end of amplification to evaluated the specificity of PCR products. The amplification efficiency of the five functional genes ranged between 85 and 93%, with the R² values ≥ 0.99.

Wang Q., Zhao Z., Yuan M., Zhang Z., Chen S., Ruan Y, & Huang Q. (2022). Impacts of urea and 3,4-dimethylpyrazole phosphate on nitrification, targeted ammonia oxidizers, non-targeted nitrite oxidizers, and bacteria in two contrasting soils. Frontiers in Microbiology, 13, 952967.

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RT-qPCR Gene Expression Analysis

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One microgram of total RNA was converted to cDNA using Superscript II reverse transcriptase and oligo-(dT)_12-18 primers (both from Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT-qPCR was performed in a 20-µl reaction mixture containing 1 µl cDNA, 10 µl SYBR Premix EX Taq (Takara Bio, Inc., Otsu, Japan), 0.4 µl 50× Rox reference dye (Takara Bio, Inc.), and 200 nM primers for each gene. The primer sequences were: NAGA (forward), 5'-CCCAAGGGTGAACTACAGTCT-3'; NAGA (reverse), 5'-GCTCCACGAACCAATTCAGGAT-3'; GAPDH (forward), 5'-AATCCCATCACCATCTTCCA-3'; and GAPDH (reverse), 5'-TGGACTCCACGACGTACTCA-3'. The reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 95℃ for 30 s, followed by 40 cycles of 95℃ for 3 s and 60℃ for 30 s, and a single dissociation cycle of 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s. All reactions were performed in triplicate, and the specificity of the reaction was determined using melting curve analysis at the dissociation stage. Comparative quantification of each target gene was performed based on the cycle threshold (C_T) normalized to GAPDH using the 2^−ΔΔCt method.

Ha Y.N., Sung H.Y., Yang S.D., Chae Y.J., Ju W, & Ahn J.H. (2017). Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 22(1), 43-51.

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Quantifying Bacterial and Archaeal Abundance

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qPCR was performed using ABI7900HT (Applied Biosystems, Foster City, CA, United States). qPCR was performed by the SYBR Green method. 16S rRNA-1369F/1492R was used to quantify total bacteria abundance, using Arch-amoA 23F/A616R and amoA-1F/2R for AOA and AOB, respectively (Rotthauwe et al., 1997 (link); Tourna et al., 2008 (link)). Plasmids containing the target fragments diluted in 10 x series were used to make qPCR standard curves. The reaction systems are all 10 μ L, including:5 μL 2× SYBR green mix II (TaKaRa Biotechnology Co. Ltd., Dalian, China), 1 μL (10 μM) of forward and reverse primers, 0.2 μL 50× Rox Reference Dye (TaKaRa Biotechnology Co. Ltd., Dalian, China), 5 ng DNA template and deionized water.
The 16S rRNA qPCR procedure was as follows: 95°C pre-denaturation for 30 s; followed by 40 cycles of 95°C denaturation for 5 s, 60°C annealing for 30 s, 72°C extension for 30 s; and then a final extension at 72°C for 1 min. The AOA and AOB amoA gene qPCR procedures were the same, except that the annealing temperature was 53°C and 55°C, respectively. Deionized water instead of soil DNA was used as a negative control to determine DNA contamination (Vestergaard et al., 2017 (link)). Three parallels were done for each sample. The results were required to have an amplification efficiency greater than 95%, R² > 0.999, and a single peak for the melting curve.

Han L., Qin H., Wang J., Yao D., Zhang L., Guo J, & Zhu B. (2023). Immediate response of paddy soil microbial community and structure to moisture changes and nitrogen fertilizer application. Frontiers in Microbiology, 14, 1130298.

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Quantitative Methylation-Specific PCR Assay

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Primers of seven candidate biomarkers (PCDH17, POU4F2, TCF21, ZNF154, EOMES, GDF15 and NID2) and ALU-C4 (reference gene) were used in the qMSP assay of the bisulfite modified DNA samples. Each reaction mixture (20 μl in total) was set to be consisted with 10 μl of SYBR^® Premix Ex TaqTM II (TAKARA), 2 μl of 10 μM mixed primers, 0.4 μl of 50 × ROX Reference Dye (TAKARA), 5.6 μl of nuclease-free water, and 2 μl of DNA template. The reactions were performed on StepOnePlus^™ Real-Time PCR System (Applied Biosystems) after a pre-incubation of 2 minutes at 95°C, and were executed for 45 cycles at 95°C for 15 seconds each, situated at 65°C for 30 seconds, followed by the extension step at 72°C for 30 seconds as one cycle. The fluorescence signal was measured at the end of each extension step at 72°C. Every reaction repeated three times. The mean of the Ct values of three times were used as the final Ct value.

Wang Y., Yu Y., Ye R., Zhang D., Li Q., An D., Fang L., Lin Y., Hou Y., Xu A., Fu Y., Lu W., Chen X., Chen M., Zhang M., Jiang H., Zhang C., Dong P., Li C., Chen J., Yang G., Liu C., Cai Z., Zhou F, & Wu S. (2015). An epigenetic biomarker combination of PCDH17 and POU4F2 detects bladder cancer accurately by methylation analyses of urine sediment DNA in Han Chinese. Oncotarget, 7(3), 2754-2764.

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Quantitative Expression Analysis of B. mori

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RNAiso Plus (Takara Dalian, China) was used to isolate total RNA from B. mori wing disc from 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3), respectively. Two μg of total RNAs from each stage was used to synthesize cDNA using reverse transcriptase (Takara) and oligo d (T) primer (Takara). The total volume of real-time quantitative PCR reactions was 20 μl, which contained 10 μl of 2 × SYBR^® Premix EX Taq™ (Takara), 0.4 μl of 50 × ROX Reference Dye (Takara) and 0.4 μl of specific primers (10 μM). Detail information of qRT-PCR Primers were provided in Additional file
4. qRT-PCR was performed with an ABI7300 real-time PCR system (Applied Biosystems, Foster City, CA), using the following condition: 95°C for 30 s followed by 40 cycles in 95°C for 5 s, 60°C for 30 s and 72°C for 31 s. The mRNA quantity of each gene was calculated with the 2^-△△Ct method and normalized to the abundance of a house-keeping gene, Bmβ-actin (GenBank NO: EU780706.1). The relative mRNA levels of each gene were represented as folds over the expression levels of Bmβ-actin.

Ou J., Deng H.M., Zheng S.C., Huang L.H., Feng Q.L, & Liu L. (2014). Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis. BMC Genomics, 15(1), 820.

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RT-qPCR Gene Expression Analysis

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One microgram of total RNA was converted to cDNA using Superscript II reverse transcriptase and oligo-(dT)_12-18 primers (both from Thermo Fisher Scientific) according to the manufacturer’s instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a 20-μL reaction mixture containing 1 μL cDNA, 10 μL SYBR Premix EX Taq (Takara Bio, Otsu, Japan), 0.4 μL 50× Rox reference dye (Takara Bio), and 200 nM of gene-specific primers. The primer sequences used in this study are listed in the Supplementary Table 2. The reactions were run on a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) at 95°C for 30 seconds, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds and a single dissociation cycle of 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds. All reactions were performed in triplicate, and the specificity of the reaction was determined using melting curve analyses at the dissociation stage. Comparative quantification of each target gene was performed based on the cycle threshold normalized to glyceraldehyde 3-phosphate dehydrogenase threshold using the 2-ΔΔCt method.

Sung H.Y., Lee J.Y., Park A.K., Moon Y.J., Jo I., Park E.M., Wang K.C., Phi J.H., Ahn J.H, & Kim S.K. (2018). Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker. Journal of Stroke, 20(3), 350-361.

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Quantitative RT-PCR Analysis of Gene Expression in HEK293 Cells

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Purified RNA samples from HEK293 cells were reverse-transcribed using RNA to cDNA EcoDry Premixes (Takara Bio Inc.). The total reaction volume was adjusted to 20 μL with RNase-free water after mixing with 100 ng cDNA, 2 μL primer sets, 10 μL 2× SYBR premix Ex Taq, and 0.4 μL 50× ROX reference dye (Takara Bio Inc.). Amplification was performed under the following cycling conditions: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 40 s. Analyses were performed in triplicate for each cDNA. The relative mRNA expression levels were calculated using the comparative threshold cycle (C_t) method with GAPDH as a control, as follows: ΔC_t = C_t (GAPDH) − C_t (target gene). The fold-change in gene expression normalized to GAPDH and relative to the control sample was calculated as 2^−ΔΔCt.

Piao H., Kim J., Noh S.H., Kweon H.S., Kim J.Y, & Lee M.G. (2017). Sec16A is critical for both conventional and unconventional secretion of CFTR. Scientific Reports, 7, 39887.

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Quantification of Nitrification and Denitrification Genes

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The forward (F) and reverse (R) primer pairs amoA-23F/amoA-616R, amoA-1F/amoA-2R, narG-571F/narG-773R, nirK-876F/nirK-1055R, nirS-cd3aF/nirS-R3cd, and nosZ-1126F/nosZ-1381R were used to quantify the ammonia monooxygenase gene of archaea (Arch-amoA) and bacteria (Bac-amoA), nitrate reductase gene (narG), nitrite reductase gene (nirK and nirS), and nitrous oxide reductase gene (nosZ). The primer sequences and thermal conditions used for the analysis are listed in Table 3. The quantitative polymerase chain reaction (qPCR) assay was performed using an ABI 7900HT (Applied Biosystems, Foster City, CA, USA). The 10 μL reaction mixtures consisted of 5 μL 2× SYBR green mix II (Takara Biotechnology Co. Ltd., Dalian, China), 0.2 μL 50× Rox Reference Dye (Takara Biotechnology Co. Ltd., Dalian, China), 0.3 μL (0.4 μmol L⁻¹) each of the F and R primers, and 5 ng sample DNA template. Standard curves were constructed using a series of 10-fold dilutions of linearized plasmids containing the target gene. Three parallel PCR replicates of all samples were performed on each plate. A melting curve analysis was conducted following the assay to verify the specificity of the amplification product. The PCR efficiency was in the range of 90–110%.

Wang L., Li K., Sheng R., Li Z, & Wei W. (2019). Remarkable N2O emissions by draining fallow paddy soil and close link to the ammonium-oxidizing archaea communities. Scientific Reports, 9, 2550.

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Real-Time PCR for Epithelial-Mesenchymal Transition Markers

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Real-time PCR was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, California, USA) using a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference. The real-time PCR reaction was performed with 2 μl cDNA, 1× TB Green^® Premix Ex Taq™ II (Tli RNase H Plus), ROX Reference Dye (TaKaRa Bio, Shiga, Japan, #RR820A), and 10 pM primers (Regular Oligo Service, cosmoGENETECH, Seoul, Korea, Table 1^{45 (link)}) under the following reaction conditions: initial step at 95 °C for 10 s, and then 40 cycles of cycling processes (95 °C for 5 s and 60 °C for 30 s).Table 1

Real-time PCR primer sets.

Gene	Protein	Primer	Sequence (5’–3’)
KCNC4	KCNC4	F	AAT ATC CCA GGG TGG TGA CA
KCNC4	KCNC4	R	GGT CTT CAA AGC TCC AGT GC
CDH1	E-cadherin	F	GAA GGT GAC AGA GCC TCT GGA T
CDH1	E-cadherin	R	GAT CGG TTA CCG TGA TCA AAA TC
CDH2	N-cadherin	F	CCT TTC AAA CAC AGC CAC GG
CDH2	N-cadherin	R	TGT TTG GGT CGG TCT GGA TG
VIM	vimentin	F	TCT ACG AGG AGG AGA TGC GG
VIM	vimentin	R	GGT CAA GAC GTG CCA GAG AC
GAPDH	GAPDH	F	CTC TGC TCC TCC TGT TCG AC
GAPDH	GAPDH	R	ACG ACC AAA TCC GTT GAC TC

Sim H.J., Kim M.R., Song M.S, & Lee S.Y. (2024). Kv3.4 regulates cell migration and invasion through TGF-β-induced epithelial–mesenchymal transition in A549 cells. Scientific Reports, 14, 2309.

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Quantitative RT-PCR Analysis of GABRP Expression

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Total RNA was extracted from the tumor metastases and SK-OV-3 cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. One microgram of total RNA was converted to cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo-(dT)_12–18 primers (Invitrogen) according to the manufacturer's instructions. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed in a 20 μl reaction mixture containing 1 μl cDNA, 10 μl SYBR Premix EX Taq (Takara Bio, Otsu, Japan), 0.4 μl Rox reference dye (50 ×, Takara Bio), and 200 nM primers for each gene. The primer sequences were: GABRP (forward), 5′-CTCGATTCAGTCCCTGCAAGA-3′ GABRP (reverse), 5′-GTGCGGGACCCGATCAT-3′ GAPDH (forward), 5′-AATCCCATCACCATCTTCCA-3′ and GAPDH (reverse), 5′-TGGACTCCACGACGTACTCA-3′. The reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) at 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s and a single cycle of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s to generate dissociation curves. All PCR reactions were performed in triplicate, and the specificity of the reaction was determined by melting curve analysis. Comparative quantification of each target gene was performed based on cycle threshold (C_t) normalized to GAPDH using the ΔΔC_t method.

Sung H.Y., Yang S.D., Ju W, & Ahn J.H. (2017). Aberrant epigenetic regulation of GABRP associates with aggressive phenotype of ovarian cancer. Experimental & Molecular Medicine, 49(5), e335-.

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