The reporter gene is a kind of gene that makes the expression product easy to detect. Its principle of action is to fuse the coding sequence of the reporter gene and its expression regulatory sequence with the target gene to form a chimeric gene, and express it under the action of the regulatory sequence. The expression status of the target gene is evaluated by detecting the reporter gene expression product.  

 

Common reporter genes include Luciferase, chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), secreted human placental alkaline phosphatase (SEAP) and green fluorescent protein (GFP), etc.

 

Luciferase is a kind of enzyme that can catalyze the oxidation and luminescence of luciferin or fatty aldehydes in organisms. It is isolated from organisms that can emit luminescence in nature. Luciferase is widely used as a reporter gene (marker protein) because of its strong specificity, high sensitivity, low endogenity, and detection is not affected by other substances in cells.

 

The double luciferase reporter gene detection system introduces an "internal reference control" reporter gene on the basis of the single luciferase reporter gene, which can minimize the influence of external factors such as cell number, cell growth status and transfection efficiency on the experiment, making the experimental results more credible.

 

In this issue, Xiaoai brings you an explosive dual luciferase reporter gene detection kit from Absin: dual luciferase reporter gene detection kit (abs60341).

 

Table 1 Dual luciferase reporter gene detection kit (abs60341)

Components	Name	100T	1000T
A	5× Passive Luciferase Lysis Buffer	10ml	100ml
B	Firefly Luciferase Assay Buffer	10ml	100ml
C	D-Luciferin	2mg	20mg
D	Renilla Luciferase Assay Buffer	10ml	100ml
E	50×Coelenterazine	200ul	2×1ml

 

1. Product features

 

Simple operation: single tube detection, firefly luciferase and Renilla luciferase are detected in the same tube;

High accuracy: The system contains Renilla fluorescein, which can correct the errors caused by the number of cells between wells, transfection efficiency, and cell growth status to ensure the accuracy of the detection results. High sensitivity and good linearity;

Good compatibility: The optical signal can be compatible with chemiluminescence instrument, multifunctional microplate reader or liquid scintillation instrument for measurement.

 

2. Product introduction

 

This kit first uses Luciferin as a substrate to detect the activity of firefly luciferase, then adds substances that inhibit the catalysis of firefly luciferase, and adds Coelenterazine to detect the activity of Renilla luciferase, realizing double luciferase reporter gene detection. It is suitable for transient transfection experiments, situations where co-reporter genes are required to establish internal references or realize multiple signals, and situations where reporter genes are required to be weakened, such as studying the cellular response of negative regulation to stimulation, and achieving higher detection accuracy.

 

Figure 1 Experimental flow of dual luciferase reporter gene detection kit

 

3. Application

 

The dual luciferase reporter gene detection kit is a widely used scientific research tool. Let me introduce the application of common luciferase reporter genes in detail with examples.

 

Application 1: Study of cis-acting elements

 

 

The double fluorescence reporter results of different promoter truncation reporter vectors of chicken bone morphogenetic protein 15 (BMP15) gene showed that the promoter of BMP15 gene was the core region-153-1 bp.

 

Application 2: Cell signaling pathway research

 

Fig. 3 Detecting the effects of different concentrations of HLY78 series derivatives on the luciferase activity of Wnt signaling pathway reporter gene TOPflash

 

By detecting the effects of different concentrations of HLY78 series derivatives on the luciferase activity of TOPflash, the reporter gene of Wnt signaling pathway, it was screened that HLY78 and HLY119 had inhibitory effects on Wnt signaling pathway.

 

Application 3: Study of trans-acting factors

 

 

Figure 5 Runx1 promotes Mrgprd gene transcription through +70 ~ +143 locus sequence

 

There may be many mechanisms for Runx1 (transcription factor) to regulate the transcription of Mrgprd gene, not just by binding to the binding site upstream of Mrgprd gene to directly regulate its transcription.

 

Application 4: miRNA target research

 

Figure 6 miRNA target research case

 

Overexpression of miR-1269b significantly reduced the luciferase activity of METTL3-WT in 293T cells, but had no significant effect on the activity of METTL3-MUT, indicating that miR-1269b can target METTL3 3 'UTR and negatively regulate METTL3 expression in GC cells.

 

Note: Application data are all from literature

 

4. Experimental data

 

Figure 7 Comparison results between Abisn dual luciferase reporter gene detection kit and similar products on the market

 

A comparative experiment was conducted with similar products on the market by using Absin dual luciferase reporter gene detection kit (abs60341), and it was found that the ratio of firefly luciferase to Renilla luciferase was closest to similar products of well-known foreign brands.

 

Partner product Xiaoai recommends this issue:

 

✔ Efficient transfection   ✔ Low toxicity   ✔ Simple operation

 

Item number	Product Name	Specifications
abs60322	Lipofect 5000 plasmid transfection reagent	0.5ml/1.0ml
abs60327	SP suspension cell plasmid DNA transfection reagent	0.5ml/1.0ml/1.5ml
abs60255	Plasmid DNA transfection kit (adherent cells)	0.5ml/1.0ml/1.5ml
abs60256	PM Primary Cell Plasmid DNA Transfection Kit	0.5ml/1.0ml/1.5ml
abs60369	RF 293 Cell DNA Transfection Kit	0.5ml/1.5ml
abs60257	Solution A (Transfection Enhancer, for plasmid DNA transfection only)	0.25ml/0.5ml/1ml

References:

[1] Zhao Haiwei, Han Peijun, Lu Xin, et al. Application of luciferase reporter gene in virology research [J]. Biotechnology Communications, 2015, 26 (1): 131-134.

[2] Nature chemical biology vol. 9,9 (2013): 579-85. doi:10.1038/nchembio.1309.

[3] Gao Guangliang, Li Hui, Wang Qigui. Analysis of Promoter Activity of Basal Fatty Acid Binding Protein Gene in Chicken Liver [C]//Representative Conference of Poultry Branch of Chinese Society of Animal Husbandry and Veterinary Medicine and National Poultry Symposium. 2013.

[4] Kang, Jian , et al. "MicroRNA-1269b inhibits gastric cancer development through regulating methyltransferase-like 3 (METTL3)." Bioengineered 12.1(2021):1150-1160.

[5] Liu Zijing, Cao Cheng. Study on the molecular mechanism of transcription factor Runx1 on Mrgprd gene transcription regulation [J]. South China Journal of National Defense Medicine, 2020, 34 (9): 5.

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.net

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