Firefly Luciferase Reporter Cell Lines for Monitoring
TF Binding and Activation

Simple, rapid, and sensitive Firefly Reporter system

Gene function analysis, target discovery and validation, assay development, and compound screening often need cell-based assays. Stable cell lines that are engineered to express a gene of interest via transgene integrations into the host genome provide an efficient approach to conduct such analysis.

At Signosis, we help researchers save time and labor in generating and validating stable cell lines so scientists can spend more effort on solving the big questions. We offer a wide selection of validated Firefly luciferase reporter cell lines that measure transcription factor activity as a read-out for various signaling pathways.

Principle

TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites. There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways. Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression. Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation

Benefits

Consistent

​TF reporter construct is stably integrated into the genome to avoid experimental/cell-to-cell variations.

Time-Saving

Cell line can be used for experiments right away to study different signaling pathways.

Routine Mycoplasma Testing

All cell lines tested negative for mycoplasma.

Signosis Firefly Luciferase Substrate

We also offer our own Firefly Luciferase Substrate which can generate a strong light signal that is comparable to our competitor’s, but at a more cost-effective price.

Signaling Pathways – Cell Lines

Gaussia Luciferase Reporter Cell Lines for Monitoring
TF Binding and Activation

Simple and Nonlethal Secreted Gaussia Luciferase Reporter system

Gene function analysis, target discovery and validation, assay development, and compound screening often need cell-based assays. Stable cell lines that are engineered to express a gene of interest via transgene integrations into the host genome provide an efficient approach to conduct such analysis.

At Signosis, we help researchers save time and labor in generating and validating stable cell lines so scientists can spend more effort on solving the big questions. We offer a wide selection of validated luciferase reporter cell lines that measure transcription factor activity as a read-out for various signaling pathways.

Principle

TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites. There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways. Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive Gaussia Luciferase gene expression.

The Gaussia Luciferase is a gene from the copepod Gaussia princeps, which is a small, naturally secreted luciferase of approximately 20kDa. This luciferase is naturally secreted from the cells, which allows for the measurement of reporter activity in the media without harming the cells.

Gaussia luciferase requires a coelenterazine-based luciferase substrate for reading and should not be used with firefly luciferase substrate.

Benefits

Naturally Secreted Luciferase

Gaussia Luciferase is naturally secreted from the cells. This also means readings can be taken without killing or lysing the cells.

Consistent

​TF reporter construct is stably integrated into the genome to avoid experimental/cell-to-cell variations.

Time-Saving

Cell line can be used for experiments right away to study different signaling pathways.

Signosis Gaussia Luciferase Substrate

We offer our own 1 step Gaussia Luciferase Substrate that is simple and easy to use while being comparable to our competitor’s substrates in price.

Products

Related Products

GAL4 Reporter Stable Cell Lines

The GAL4-UAS system is a powerful technique for studying gene expression and function. It has also been adapted to study nuclear receptor chemical-binding functions. Signosis GAL4-UAS-luc Reporter Stable Cell Lines is a reliable, rapid, and sensitive method for analyzing the expression of your target gene upon exposure to related ligands or other stimuli.

Principle

Nuclear hormone receptors (NHRs) are a group of ligand-binding transcription factors (TFs). More than 350 NHRs are available in the protein data bank. Like other TFs, they can regulate gene expression by binding to specific DNA regulatory elements, but their activities are modulated by the corresponding ligands.

Although DNA regulatory element-luciferase reporter cell lines can be used to study endogenous or exogenous receptors and analyze the receptor signaling pathway in a native biological context, the NHR DNA element-luciferase reporter cell lines lack the specificity and uniqueness due to the similarity in binding sequences among their entire family members.

Signosis has developed ligand binding domain(LBD) -driven GAL4 reporter stable cell lines by delivering two vectors, one expressing 8 copies of GAL4 upstream activator sequences (UAS) in front of the luciferase reporter gene, another expressing Gal4 DNA Binding Domain (DBD) bound to a ligand binding domain (LBD) of your choice. Upon addition of a corresponding ligand, the DBD-LBD fusion protein is activated, which binds to GAL promoter binding site that will drive forward the expression of luciferase. With this method, our GAL4 based cell line assays will have minimal cross-reactivity with other nuclear receptor, leading to the assays having high sensitivity and specificity, while also having low toxicity of the chimeric receptors to the cells when overexpressed. These cell lines can be used to screen drug compounds for agonist or antagonist.

​Stable clones were selected with both hygromycin and G418, and a functional assay was subsequently conducted where clones with the highest induction fold chosen.

Benefits

Less Toxic

The chimeric receptors display less toxic effects on the cells.

Highly Sensitive

More than 50 fold induction in response to the corresponding stimuli.

Routine Mycoplasma Testing

All cell lines tested negative for mycoplasma.

Signosis Firefly Luciferase Substrate

We also offer our own Firefly Luciferase Substrate which can generate a strong light signal that is comparable to our competitor’s, but at a more cost-effective price.

NR LBD-Driven GAL4 Cell Lines

Signosis has developed ready-to-use Nuclear Receptor Ligand Binding Domain driven GAL4 reporter cell lines.

These cell lines are a reliable, rapid, and sensitive method for analyzing the intracellular status of NRs upon exposure to drug candidates or other stimuli. These functional assays allow for measurement of receptor activation or inhibition by compounds and can be used to determine compound potency and selectivity.

Ultra Sensitive NR LBD-DRIVEN GAL 4 Cell Lines

The Ultra Sensitive LBD-driven GAL4 reporter cell lines has been optimized with an enhanced promoter upstream of the luciferase gene to maximize signal output in response to extremely low concentrations of inducers. These cell line not only yields the enhanced basal signal suitable for any instrument detection, but also produces the higher induction to monitor the specific pathways in an ultra sensitive manner.

GAL4-UAS-Luc Cell Lines

Signosis has developed the GAL4-UAS-Luc Reporter Stable Cell Lines as a reliable, rapid, and sensitive method for analyzing the expression of your target gene of interest upon exposure to related ligands or other stimuli. These cell lines are transfected only with the GAL4-UAS-LUC vector, and are ready to be transfected with your customized GAL4 vector containing your NR of interest.

Research Application

GAL4 LBD-driven GAL4 Reporter HEK 293

• Studying the effects of ligand binding on GAL4-mediated transcriptional activity
• Identifying ligands that activate or inhibit the target receptors by monitoring GAL4-driven luciferase expression
• Screening for potential therapeutic compounds that modulate the target receptors by measuring changes in luciferase activity
• Investigating the mechanisms underlying the regulation of the target receptors by analyzing the GAL4-mediated transcriptional response to various stimuli
• Comparing the activity and selectivity of different compounds on the target receptors by using the GAL4 reporter assay
• Studying the interaction between the target receptors and other signaling pathways or co-regulators by analyzing the changes in GAL4-mediated transcriptional activity upon co-expression of different genes or treatments.

PPAR-gamma LBD-driven GAL4 reporter HEK 293

• Studying the effects of ligand binding on PPAR-gamma-mediated transcriptional activity
• Identifying compounds that activate or inhibit PPAR-gamma activity by monitoring GAL4-driven luciferase expression
• Investigating the role of PPAR-gamma in adipogenesis and glucose homeostasis by analyzing the GAL4-mediated transcriptional response to various stimuli
• Screening for potential therapeutic compounds that target PPAR-gamma for the treatment of diabetes and other metabolic disorders
• Studying the interaction between PPAR-gamma and other signaling pathways or co-regulators by analyzing the changes in GAL4-mediated transcriptional activity upon co-expression of different genes or treatments.

PPAR-alpha and PPAR-delta LBD-driven GAL4 reporter HEK 293

• Studying the effects of ligand binding on PPAR-alpha or PPAR-delta-mediated transcriptional activity
• Identifying compounds that activate or inhibit PPAR-alpha or PPAR-delta activity by monitoring GAL4-driven luciferase expression
• Investigating the roles of PPAR-alpha and PPAR-delta in lipid metabolism and energy homeostasis by analyzing the GAL4-mediated transcriptional response to various stimuli
• Screening for potential therapeutic compounds that target PPAR-alpha or PPAR-delta for the treatment of metabolic disorders, including dyslipidemia and obesity
• Studying the interaction between PPAR-alpha or PPAR-delta and other signaling pathways or co-regulators by analyzing the changes in GAL4-mediated transcriptional activity upon co-expression of different genes or treatments.

Related Products

Receptor Tyrosine Kinase & HER2, Mutants Stable Cell Line

Gene function analysis, target discovery and validation, assay development, and compound screening often need cell-based assays. Stable cell lines that are engineered to express a gene of interest via transgene integrations into the host genome provide an efficient approach to conduct such analysis.

At Signosis, we help researchers save time and labor in generating and validating stable cell lines so scientists can spend more effort on solving the big questions. We offer a wide selection of validated luciferase reporter cell lines that measure transcription factor activity as a read-out for various signaling pathways.

Principle

BaF3, a murine interleukin-3 dependent pro-B cell line has been well-known as a model system for assessing downstream signaling of kinase oncogenes, and the ability of small-molecule kinase inhibitors to block kinase activity. Even though BaF3 is suitable as a transfection or transduction host, the viral delivery system is still the main approach. Signosis has been engineered as a non-viral vector-based electroporation delivery system to establish a panel of EGFR, Flt3 and Her2 and mutants BaF3 stable cell lines. The cell lines have been primarily selected with GFP, and subsequently validated with PCR-sequencing and finally confirmed western blot. The clone with the strongest expression level has been launched for facilitating drug screening and associated researches.

Benefits

Double Selection

​The single clones have been selected with GFP and hygromycin.

Solid Validation

The selected clones have been validated with PCR-sequencing and Western blot analysis.

Functional Analysis

​The cell lines have been conducted the proliferation test with the corresponding inhibitors.

Receptor Kinase & Mutant Stable Cell Line

EGFR Mutants

EGFR Stably Expressing Cell Lines

EGFR mutations occur in many types of cancers including non-small cell lung cancer. Common mutations found in these patients are deletions in exon 19 (Del19) and L858R substitution in exon 21. Patients with these mutations are sensitive to first (gefitinib or erlotinib) and second (afatinib) generation EGFR tyrosine kinase inhibitors (TKIs), whereas patients with wild type EGFR are not. However, eventually, all patients become resistant to first and/or second-generation EGFR TKIs, and acquired T790M secondary mutation in exon 20 accounts for more than 50% of the acquired resistance. Cells expressing EGFR with either L858R+ T790M or Del19+T790M double mutations are resistant to induced apoptosis in the presence of first or second-generation EGFR TKIs.

Eight cell lines stably expressing HA-tagged EGFR have been established in the IL3-dependent Ba/F3 cell lines —WT EGFR, L858R EGFR mutant, L858R/T790M EGFR double mutant, Del19 (del747-752) EGFR mutant, Del19/T790M EGFR mutant, three ins20 (D770_N771insSVD, A763_Y764insFQEA, A767_dupASV) EGFR mutants —and Ba/F3 cell line expressing empty vector. Also, two cell lines stably expressing HA-tagged EGFR have been established in HCC827 cell lines —Exon19del EGFR mutant, Exon19del/T790M EGFR double mutant—and HCC827 cell line expressing empty vector.

Due to their proliferation upon EGFR activation, sensitivity to TKIs, or resistance to induced apoptosis, these cell lines can be used to study the molecular mechanism underlying susceptibility of tumors to the drugs as well as screening and validating new TKIs.

HER2 Mutants

HER2 (ERBB2) Stably Expressing Cell Lines
​

The proto-oncogene human epidermal growth factor receptor 2 (HER2) gene also known as ERBB2 is a member of the ERBB/HER RTK family which is comprised of EGFR (EGFR/HER1/ERBB1), HER3/ERBB3, and HER4/ERBB4. This gene encodes for a transmembrane glycoprotein receptor with an intracellular domain with tyrosine kinase activity which is responsible for the activation of intracellular signal transduction pathways controlling epithelial cell growth, differentiation, and angiogenesis. Aberrations of the HER2 gene which usually leads to increased receptor signaling have been observed in many different types of cancer.

FLT3 Mutants

FLT3 Stably Expressing Cell Lines

The FMS-like tyrosine kinase 3 (FLT3) also known as cluster of differentiation antigen 135 (CD135) or fetal liver kinase-2 (Flk2) is a class III receptor tyrosine kinase (RTK) that plays a key role in controlling survival, proliferation, and differentiation of hematopoietic cells and it widely expresses in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Mutations in FLT3 (both internal tandem duplications (ITD) and point mutations) are among the most common somatic mutations in AML patients and are associated with poor prognosis and outcome in those patients

Research Applications

EGFR mutants

• Investigating the effects of EGFR mutations on downstream signaling pathways and cellular processes, such as cell proliferation, differentiation, and survival.
• Identifying novel therapeutic strategies for targeting EGFR mutants in cancer by testing the efficacy of various inhibitors or combination therapies.
• Screening for small molecule compounds or antibodies that selectively target EGFR mutants over wild-type EGFR.
• Developing in vitro and in vivo models to study the mechanisms of resistance to EGFR inhibitors and to test novel combination therapies.
• Evaluating the potential of EGFR mutants as diagnostic and prognostic biomarkers for specific types of cancer.

HER2 (ERBB2) and mutants

• Investigating the effects of HER2 mutations on downstream signaling pathways and cellular processes, such as cell proliferation, differentiation, and survival.
• Identifying novel therapeutic strategies for targeting HER2 and its mutants in cancer by testing the efficacy of various inhibitors or combination therapies.
• Screening for small molecule compounds or antibodies that selectively target HER2 mutants over wild-type HER2.
• Developing in vitro and in vivo models to study the mechanisms of resistance to HER2 inhibitors and to test novel combination therapies.
• Evaluating the potential of HER2 and its mutants as diagnostic and prognostic biomarkers for specific types of cancer.

FLT3 and mutants

• Investigating the effects of FLT3 mutations on downstream signaling pathways and cellular processes, such as cell proliferation, differentiation, and survival.
• Identifying novel therapeutic strategies for targeting FLT3 and its mutants in cancer by testing the efficacy of various inhibitors or combination therapies.
• Screening for small molecule compounds or antibodies that selectively target FLT3 mutants over wild-type FLT3.
• Developing in vitro and in vivo models to study the mechanisms of resistance to FLT3 inhibitors and to test novel combination therapies.
• Evaluating the potential of FLT3 and its mutants as diagnostic and prognostic biomarkers for specific types of cancer.

Baf3 Stable Cell Lines

• Studying the signaling pathways and cellular processes that are regulated by specific receptors or mutants expressed in the Baf3 stable cell lines.
• Identifying and characterizing novel ligands or compounds that modulate the activity of the receptors or mutants expressed in the Baf3 stable cell lines.
• Evaluating the efficacy and selectivity of various inhibitors or combination therapies targeting the receptors or mutants expressed in the Baf3 stable cell lines.
• Developing in vitro and in vivo models to study the effects of specific mutations or treatments on cell proliferation, differentiation, and survival in the context of the Baf3 stable cell lines.
• Investigating the mechanisms of resistance to various inhibitors or combination therapies in the Baf3 stable cell lines, and testing novel strategies to overcome resistance.

EGFR Vectors

pMSCV-EGF-GFP Vectors

pCMV-EGFR Vectors

pLenti-MSCV-EGFR-GFP Vectors

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that plays a critical role in regulating cell division and death. Mutations in EGFR gene that lead to overexpression of the protein have been associated with a number of different cancers. The EGFR gene is present on chromosome 7p11.2 and has 28 coding exons in which exons 5–7 and 13–16 code for the ligand-binding domain and exons 18–24 code for the TK domain.

Downstream effects of EGFR activation include modulation of three major pathways of RAS-RAF-MAPK, PI3K/AKT, and JAK/STAT pathways which can lead to cell survival, proliferation, and migration of cancer cells.

​We offer a collection of unique cloning and expression cDNA vectors contain WT and mutated EGFR gene. These mutations are clinically approved and are under further investigation by many researchers. Our high-quality vectors are fully sequenced and are ready to use. We also offer custom cloning services for your specific construct needs.
​

pMSCV-EGF-GFP Vectors Research Application

• ​Overexpressing EGF in cells to study the effects of EGF on cell proliferation, differentiation, and survival.
• Generating stable cell lines expressing EGF for long-term studies of EGF signaling pathways and downstream effects.
• Investigating the mechanisms of resistance to EGF inhibitors or combination therapies in cells that overexpress EGF.
• Developing animal models of EGF-driven cancers by transfecting cells with pMSCV-EGF-GFP vectors and injecting them into animals.
• Evaluating the efficacy of various inhibitors or combination therapies targeting EGF in vitro and in vivo.

pCMV-EGFR Vectors

Benefits

DYKDDDDK-Tag for Easy Detection

This tag is engineered to downstream of EGFR gene to facilitate Western blot.

Perfect for Stable Cell Line Establishment

The hygromycin-resistant gene is embedded in the vector for stable cell line establishment.

Custom Service Available

We also offer the custom service with the same list price. If you have any mutants in your mind, please let us know.

pLenti-MSCV-EGFR-GFP Vectors

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that plays a critical role in regulating cell division and death. Mutations in EGFR gene that lead to overexpression of the protein have been associated with a number of different cancers. The EGFR gene is present on chromosome 7p11.2 and has 28 coding exons in which exons 5–7 and 13–16 code for the ligand-binding domain and exons 18–24 code for the TK domain. Downstream effects of EGFR activation include modulation of three major pathways of RAS-RAF-MAPK, PI3K/AKT, and JAK/STAT pathways which can lead to cell survival, proliferation, and migration of cancer cells.

​We offer a collection of unique cloning and expression cDNA vectors contain WT and mutated EGFR gene. These mutations are clinically approved and are under further investigation by many researchers. Our high-quality vectors are fully sequenced and are ready to use. We also offer custom cloning services for your specific construct needs.

pCMV-EGFR Vectors Research Application

• ​Overexpressing EGFR in cells to study the effects of EGFR on cell proliferation, differentiation, and survival.
• Generating stable cell lines expressing EGFR for long-term studies of EGFR signaling pathways and downstream effects.
• Investigating the mechanisms of resistance to EGFR inhibitors or combination therapies in cells that overexpress EGFR.
• Developing animal models of EGFR-driven cancers by transfecting cells with pCMV-EGFR vectors and injecting them into animals.
• Evaluating the efficacy of various inhibitors or combination therapies targeting EGFR in vitro and in vivo.

pLenti-MSCV-EGFR-GFP Vectors

Benefits

Super bright CopGFP under EF-1 promoter as a selection marker

Contain HIV-1 LTRs and the lentiviral packaging signal (Ψ)

Specific elements (WPRE, cPPT, RRE) included for improving transgene expression

pLenti-MSCV-EGFR-GFP Vectors

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that plays a critical role in regulating cell division and death. Mutations in EGFR gene that lead to overexpression of the protein have been associated with a number of different cancers. The EGFR gene is present on chromosome 7p11.2 and has 28 coding exons in which exons 5–7 and 13–16 code for the ligand-binding domain and exons 18–24 code for the TK domain. Downstream effects of EGFR activation include modulation of three major pathways of RAS-RAF-MAPK, PI3K/AKT, and JAK/STAT pathways which can lead to cell survival, proliferation, and migration of cancer cells.

​

We offer a collection of unique cloning and expression cDNA vectors contain WT and mutated EGFR gene. These mutations are clinically approved and are under further investigation by many researchers. Our high-quality vectors are fully sequenced and are ready to use. We also offer custom cloning services for your specific construct needs.

pLenti-MSCV-EGFR-GFP Vectors Research Application

• ​Generating stable cell lines expressing EGFR for long-term studies of EGFR signaling pathways and downstream effects.
• Investigating the effects of EGFR mutations on downstream signaling pathways and cellular processes, such as cell proliferation, differentiation, and survival.
• Identifying novel therapeutic strategies for targeting EGFR in cancer by testing the efficacy of various inhibitors or combination therapies.
• Screening for small molecule compounds or antibodies that selectively target EGFR mutants over wild-type EGFR.
• Developing animal models of EGFR-driven cancers by transfecting cells with pLenti-MSCV-EGFR-GFP vectors and injecting them into animals.
• Evaluating the potential of EGFR and its mutants as diagnostic and prognostic biomarkers for specific types of cancer.

Benefits

Super bright CopGFP under EF-1 promoter as a selection marker

DYKDDDDK-tag engineered to downstream of EGFR gene for Western blot

Parental Cell Lines

Signosis offers some of the parental (Host) cells used for generating its stable cell lines. These cell lines can be used as a matched control for their related stable cell lines. All cell lines tested negative for mycoplasma.

Principle

A parental cell line is a group of cells that are derived from the same source or organism and are used as a starting point for creating different cell lines. Parental cell lines are often used in biomedical research and drug development to establish a baseline or control group for comparison purposes.

​Parental cell lines can also be used to produce biologics, such as monoclonal antibodies or recombinant proteins, which are used as therapeutics. In this case, the parental cell line serves as the foundation for the production of these products.

Benefits

Consistent

Exogenous gene is stably integrated into the genome to avoid experimental/cell-to-cell variations

Cell Lines

Research Application

HeLa cells are a commonly used cell line in cancer research, derived from cervical cancer cells. They are an immortal cell line, meaning they can divide indefinitely. HeLa cells have been extensively studied and used in many different research applications due to their rapid growth, adaptability, and ability to easily transfect with exogenous DNA.

Research applications:

• Studying the mechanisms of cancer development and progression.
• Screening for novel cancer therapies.
• Evaluating the efficacy of various cancer treatments, such as chemotherapy, radiation, and immunotherapy.
• Investigating the effects of viral infections, such as HPV, on cancer development.
• Studying the regulation of cell cycle and apoptosis.

HEK293 cells are a widely used human embryonic kidney cell line. They are also an immortal cell line and can be easily transfected with exogenous DNA. HEK293 cells have been extensively used in research for protein expression and biochemical studies due to their high transfection efficiency and robust protein expression.

​Research applications:

• Protein expression and purification for biochemical and structural studies.
• Studying signaling pathways and protein-protein interactions.
• Developing recombinant viral vectors for gene therapy.
• Developing and optimizing cell-based assays for drug screening.
• Studying the effects of gene mutations on protein function.

A549 cells are a human lung carcinoma cell line commonly used in respiratory research. They were originally isolated from a patient with adenocarcinoma and have been extensively studied for their ability to differentiate into respiratory epithelial cells.

Research applications:

• Studying respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer.
• Investigating the effects of environmental toxins, such as cigarette smoke, on lung function.
• Developing and optimizing cell-based assays for drug screening.
• Studying the regulation of inflammation and immune response in the respiratory system.
• Studying the mechanisms of viral infections, such as influenza and respiratory syncytial virus (RSV).

NIH/3T3 cells are a mouse embryonic fibroblast cell line. They are widely used in research for their ability to rapidly divide and their ease of transfection with exogenous DNA.

​Research applications:

• Studying the regulation of cell cycle and apoptosis.
• Studying the effects of gene mutations on protein function.
• Developing and optimizing cell-based assays for drug screening.
• Studying the mechanisms of cancer development and progression.
• Studying the regulation of cellular metabolism and energy production.

Jurkat cells are a human T lymphocyte cell line commonly used in immunology research. They were originally isolated from a patient with acute T cell leukemia and have been extensively studied for their role in immune function.

​Research applications:

• Studying the regulation of immune function, including T cell activation, differentiation, and apoptosis.
• Studying the mechanisms of immune-related diseases, such as autoimmune disorders and cancer.
• Developing and optimizing cell-based assays for drug screening.
• Studying the effects of gene mutations on immune function.
• Studying the regulation of cellular metabolism in immune cells.

K562 cells are a human erythroleukemia cell line commonly used in cancer research. They were originally isolated from a patient with chronic myelogenous leukemia and have been extensively studied for their role in erythropoiesis and differentiation.

​Research applications:

• Studying the regulation of erythropoiesis and differentiation.
• Studying the mechanisms of cancer development and progression.
• Developing and optimizing cell-based assays for drug screening.
• Studying the effects of gene mutations on protein function.
• Studying the regulation of cellular

COS-7 is a cell line derived from the African green monkey kidney cells, commonly used in molecular biology and biotechnology research. The cells are highly transfectable, allowing for the introduction of exogenous genes and expression of recombinant proteins.

Research applications:

1. Protein expression and purification: COS-7 cells are commonly used for the production of recombinant proteins due to their high transfection efficiency and ability to express high levels of proteins.
2. Gene expression studies: COS-7 cells are often used to study the function and regulation of genes, as they can be easily transfected with plasmids containing reporter genes and other genetic constructs.
3. Viral vector production: The high transfection efficiency of COS-7 cells makes them useful for the production of viral vectors for gene therapy and other applications.
4. Drug discovery: COS-7 cells can be used in high-throughput screening assays to identify potential drug candidates for various diseases.

MCF7 is a breast cancer cell line derived from a human patient with metastatic breast adenocarcinoma. The cells are widely used in breast cancer research as a model system for studying the molecular mechanisms underlying the disease.

​Research applications:

1. Breast cancer biology: MCF7 cells are used to study various aspects of breast cancer biology, including the mechanisms of tumor progression, drug resistance, and metastasis.
2. Drug discovery: MCF7 cells are used in drug discovery research to screen for potential therapeutic compounds that may be effective in treating breast cancer.
3. Hormone receptor signaling: MCF7 cells express estrogen receptors and are used as a model system to study the mechanisms of hormone receptor signaling in breast cancer.
4. Gene expression studies: MCF7 cells are commonly used to study the expression and regulation of genes associated with breast cancer.

Associated Products

Benefits

Simple, easy to use One-step solutions

Ready to use without any prior preparation or mixing needed

Compatible

Works with all Signosis luciferase products and third-party firefly luciferase systems

Cost Effective

Less $ spent per reaction compared to our competitors

EGFR Vectors

Principle

Mutations in the epidermal growth factor receptor (EGFR) gene are commonly observed in many type of cancers including lung, colorectal, head and neck and breast cancer. These mutations play an important role in particular in non-small-cell lung cancer as they can be not only therapeutic target but also a predictive tool for response to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. Cell lines have played crucial roles in the identification and characterization of these driver mutations.

Signosis has developed expression vectors for the most common EGFR mutations harboring FLAG marker with high expression suitable for your cancer research. We also accept any other customized EGFR mutation for your research need, contact us for more information.

Benefits

DYKDDDDK-Tag for Easy Detection

This tag is engineered to downstream of EGFR gene to facilitate Western blot

Perfect for Stable Cell Line Establishment

The hygromycin-resistant gene is embedded in the vector for stable cell line establishment.

Custom Service Available

​We also offer the custom service with the same list price. If you have any mutants in your mind, please let us know.

Cell Vialbility Assay (CVC)

Principle

Cell Viability/Cytotoxicity (CVC) Reagent is a colorimetric WST-8 based assay that provides a simple and accurate method to measure cell proliferation. This is a tetrazolium reduction assays based on the cleavage of the tetrazolium salt WST-8 to water-soluble, orange formazan dye by cellular mitochondrial dehydrogenases (Fig.1). Intensity of dye is a direct proportion of increased activity of the mitochondrial dehydrogenases and therefore number of viable cells which can be quantified by measuring the absorbance at 450 nm.

Ready to use CVC Reagent allow for rapid, sensitive and reliable method for measuring cell viability/cytotoxicity directly from a 96-well plate without any additional processing.

TF Activation Plate Arrays

Profile the activation for up to 192 TFs at one time

Principle

Signosis’ TF Activation Profiling Arrays are used for monitoring the activities of multiple TFs simultaneously. In the technology, a series of biotin-labeled probes are made based on the consensus sequences of TF DNA-binding sites. When the probe mixtures incubate with nuclear extracts, individual probes will find their corresponding TFs and form TF/probe complexes, which can be easily separated from free probes through a simple spin column purification.

The bound probes are then detached from the complexes and analyzed through hybridization with a plate, which each well is specifically pre-coated with complementary sequences of the probes.

Benefits

Multiplex

​One assay allows the measurement of the activities for various transcription factors.

Simple Procedures

Use simple procedures similar to ELISA & use standard laboratory instrument, Lumniex is not needed

Works with Mammalian Nuclear Extracts

Samples can be taken from human, rat, and mouse

TF Profiling Plate Array

Profile the activation for up to 192 TFs at one time.

FewCell TF Plate Arrays

Profile the activation of TFs with limited amount of cells.

Functional Plate Arrays

Profile the activation of TFs for a specific pathway.

Promoter-Binding Profiling Plate Arrays

Profile the activation for up to 192 TFs at one time

Principle

Promoter-binding TF profiling plate arrays are a competition assay of Signosis’ TF activation plate arrays. In the TF activation plate arrays, if all of 48 or 96 targeted transcription factors exist in the assayed samples, they will form 48 or 96 types of complexes, each TF with its corresponding biotin-labeled oligo (just like the complex in the gel shift assay). After a simple spin separation of the complexes from unbound free biotin-labeled oligos with a membrane-based column, TF-bound oligos eluted from the column and used for plate hybridization in which a complementary DNA of biotin-labeled oligo. The captured oligo is then detected with streptavidin-HRP and a chemiluminescent substrate. If any TF is not present, it will not form a complex, leading to no detection of TF in the following plate assay of biotin-labeled oligo. In promoter-binding TF profiling arrays, PCR fragment containing the promoter of your interest is mixed with a set of 48 or 96 biotin-labeled oligos corresponding to 48 or 96 TFs along with an assayed sample. If unlabeled promoter DNA fragment contains a TF binding sequence, it will compete with the biotin-labeled oligo to bind to the TF in the sample, leading to no or less biotin labeled TF/DNA complex formation and no or lower detection. Through comparison in the presence and absence of the competitor promoter DNA fragment, promoter-bound TFs can be identified.

Benefits

Multiplex

​A single assay allows the characterization of binding of 48 or 96 TFs to a specific promoter

Simple Procedures

Probe incubation, spin column separation, plate hybridization, and HRP detection

TF Interaction Plate Arrays

Monitor TF/TF or TF/coregulator interactions among 48 or 96 different TFs simultaneously in relation to one TF or coregulator of interest.

Principle

Signosis’ TF-TF Interaction Plate Array can simultaneously profile the transcriptional interaction of multiple TFs with a TF or co-regulator of interest. In this assay, a series of unique biotin-labeled probes are provided that correspond with the consensus sequences of individual TF DNA-binding sites. Therefore, each probe represents an individual TF.

When the probe mix is incubated with nuclear extract, individual probes bind their corresponding TF. The TF or co-regulator of interest is then immunoprecipitated, along with transcriptionally interacting TFs, using a corresponding antibody and protein G or A agarose beads in a tube. Unbound probes and proteins are washed away. The bound probes are then detached from the complex and are subsequently denatured. The biotin-labeled DNA strands are hybridized on a pre-coat plate and detected with streptavidin-HRP and substrate. The detected signals reflect the interacting TFs with the particular TF or co-regulator. Luminescence is reported as relative light units (RLUs) on a microplate luminometer.

Benefits

Multiplex

​A single assay allows the characterization of binding of 48 or 96 TFs to a specific promoter

Quantitative Comparison

​Differences between samples can be quantitatively compared and analyzed.

No Special Equipment Required

Compatible with common chemiluminescence plate readers

Simple Procedures

Co-IP of TF complexes with magnetic beads is quick, easy, and effective.

EMSA

100+ non-radioactive EMSA Kits are available. The probes are pre-labeled with biotin, and all reagents are included in the kit.

Principle

When one TF is activated, it binds to a biotin-labeled probe, which is designed based on the consensus binding sequence of the TF. When the formed complex is subjected to electrophoresis, it runs slower than the free probe, and can be easily distinguished. Two or more samples can be compared and differences can be determined by the density of the shifted band.

Benefits

Isotope Free

It is a chemiluminescent-based detection.

No Probe Preparation

The probes included are pre-labeled and optimized.

Highly Sensitive

With optimized conditions, the kit sensitivity is compatible with that from radioactive-based detection method.

Simple Procedures

All of the reagents are included in the kit.

TF Filter Plate Arrays

This “EMSA on a plate” assay allows you to monitor the activation of a TF in vitro in up to 96 samples at one time.

Principle

In Signosis’ TF filter plate assays, a specific biotin-labeled TF DNA binding sequence is mixed with nuclear extracts to allow formation of TF-DNA complexes. A filter plate is used to retain bound DNA probe and remove free probe. The bound pre-labeled DNA probe is then eluted from the filter and collected for quantitative analysis through DNA plate hybridization. The captured DNA probe is further detected with streptavidin-HRP. Luminescence is reported as relative light units (RLUs) on a microplate luminometer.

Benefits

High Throughput Screen

A specific TF in multiple samples can be analyzed.

Flexible

Can analyze 1 to 96 samples simultaneously. Easy to reseal and store for later use.

Quantitative Comparison

TF activity differences between two or more samples can be quantitatively analyze

Wide Selection

100+ popular TFs Kits are available from Signosis.

TF ELISA Kits

TF ELISA Kit allows High-throughput profiling TF-DNA binding activity.

Principle

TF ELISA Kit is high sensitive and specific assay with a simple and optimized procedure. The 96-well (8×12 strip) clear plate is pre-immobilized with the TF consensus sequencing oligo. The activated TF in nuclear extract or the while cell lysate is added in the well and binds to the oligo. The activated TF is detected with a specific antibody against the subunit and a HRP conjugated secondary antibody. The assay utilizes colorimetric detection method, which can be easily measured by spectrophotometry.

Benefits

Positive Protein Control Included

The kit contains an active recombinant protein for a positive control.

Highly Effective

Distinguishing two pathways in one ELISA assay.

Highly Specific

Specifically monitor TF activation both the DNA binding sequence and gene specific antibody against the specific gene.

Snapshot Magnetic Beads ChIP Assays

Analyze the interaction of transcription factors and associated coactivitors and corepressors on target promoters in 10^6 cells.

Principle

The magnetic beads ChIP assay typically comprises four steps: (1) cross-linking proteins to DNA; (2) chromatin fragmentation; (3) protein precipitation; and (4) target identification and quantitation. The initial cross-linking is to insure that protein-DNA complexes remain associated through the subsequent steps. Formaldehyde is the most commonly used reagent for the cross-linking of proteins that are directly bound to DNA, such as transcription factors and histones, but not for coactivators and corepressors indirectly associated with DNA due to short spacer arm.

Several improvements in Signosis’ ChIP assay are highlighted as follows: firstly, the dual cross-linking, formaldehyde (short space arm) and second cross-linking reagent (long space arm), is introduced to ensure the detection of both TFs and associated cofactors. Secondly, DNA is broken down to mononucleosomes and 500bp in size by MNase digestion, which enhances greatly the sensitivity and reproducibility. The cross-linked protein-DNA complexes are subsequently pulled down by a specific antibody and ChIP grade protein A/G magnetic beads. The last improvement is to release DNA from cross-linked proteins with one-step chelex-100 without tedious proteinase K digestion and further purification, significantly increasing the assay efficiency.

Benefits

High Reproducibility

Controllable MNase digestion for DNA fragmentation instead of sonication.

Highly Specificity

The cross-linked protein-DNA complexes are subsequently pulled down by a specific antibody and ChIP grade protein A/G magnetic beads.

Simple Procedure

Use one step Chelex-100 treatment to release cross-linked DNA instead of tedious proteinase K digestion and column purification.

High-Throughput

Kit comes with everything you will need to perform the assay, including crosslinkers and enzyme. Dual cross linking to analyze both transcription factors and the associated factors.

TF Luciferase Vectors

Measure TF activation in cells. 100+ vectors are available. We also provide custom service to create a vector for any unlisted TFs.

pTF-Luc reporter vectors are a series of firefly luciferase-based reporter constructs for quantitative measurement of the activities of transcription factors (TFs) in cells. Each vector contains a cis-element (DNA binding sequence), a minimal promoter, and firefly luciferase gene. When a TF is activated, it binds to cis-element and results in induction of luciferase gene expression. Therefore, luciferase activity represents the activity of the TF.

Principle

The Luciferase Reporter Vectors have been specially constructed to report the binding activity of a single TF. Multiple copies (at least four) of the cis-acting enhancer element have been inserted into each vector upstream of a minimal TA promoter. Upon vector transfection and cell stimulation, the activated TF is bound to the enhancer element and initiate the transcription of downstream luciferase reporter gene. The chemiluminescent measurement of luciferase activity is proportional to the amount of expressed enzyme and thus the binding activity of the targeted TF.

Benefits

Functional Assay

Monitor the transactivation of transcription factors in cells.

Quantitative Analysis

Determine TF activation by quantitative measurement ofluciferase activity.

Stable Cell Lines

​The vectors can be used for establishing stable cells expressing the reporters.

Firefly Luciferase Substrate

Firefly Luciferase Substrate for use with the reporter vectors.

Cytokine ELISA Plate Arrays

Cytokines and growth factors are signaling molecules that have critical roles in many biological processes such as cellular growth, differentiation, gene expression, migration, immunity and inflammation. Disruption in these signals can lead to a variety of diseases, including arthritis, liver disease, inflammatory bowel disease, cardiac-related diseases, and cancers.

Signosis provides a number of tools to profile one specific cytokine or growth factor, or to monitor multiple simultaneously. See our ELISA biomarker kit.

Principle

In this assay, the test sample initially reacts with the solid phase capture antibody, resulting in the cytokine being bound to the well. The wells are then washed to remove unbound proteins, and biotin-linked antibodies are added to bind to the cytokines. After washing away the unbound antibodies, Streptavidin-HRP conjugate is added to form a complex with the antibody-bound cytokines.

​For colorimetric kits, the HRP substrate, TMB, is added and forms a blue color when the HRP-linked antibodies are detected. The reaction is then terminated with Stop Solution, which changes the color from blue to yellow. The cytokine concentration in each well is directly proportional to its color intensity and can be quantified by measuring its optical density at 450 nm (OD450) in a microplate reader.

​For chemiluminescence kits, the plate is detected with HRP luminescent substrate. Luminescence is reported as relative light units (RLUs) on a microplate luminometer. The level of expression for each specific cytokine is directly proportional to the luminescent intensity.

Benefits

High Sensitivity & Responsiveness

Requires minimal amount of sample. Compatible with common plate readers. Simple procedure with reagents included.

Multiplex

​One pre-coated plate can measure 48 cytokines for 2 human samples, 32 cytokines for 3 human samples; 32 cytokines for 3 mouse samples, 24 cytokines for 4 mouse samples; or 16 cytokines for 6 rat samples.

Quantitative Profiling

Allows quantitative comparison of expression among samples

Wide Sample Type Selection

The assay is suitable for any biological liquid material and cell lysate, such as sera, cerebrospinal fluid (CSF), urine and tissue homogenate.

Rat Cytokine ELISA Plate Array

Profile an array of rat cytokines using our ELISA kit

Mouse Cytokine ELISA Plate Array

Profile an array of mouse cytokines using our ELISA kit

Human Cytokine ELISA Plate Array

Profile an array of human cytokines using our ELISA kit

Research Discovery Objectives

Drug Screening and Discovery

Stable cell lines expressing a specific receptor or target protein can be used to screen for potential drug candidates and evaluate their efficacy and toxicity.

Protein Production

Stable cell lines can be used to produce recombinant proteins for various applications, including biopharmaceutical production, biochemical assays, and structural biology studies.

Gene Editing and Gene Therapy

Stable cell lines can be used to study gene function and develop gene editing or gene therapy approaches for genetic diseases.

Disease Modeling

Stable cell lines can be used to model genetic diseases and study their pathogenesis and potential therapeutic targets.

Vaccine Development

Stable cell lines can be used to produce recombinant proteins for vaccine development and production.

Cell-Based Assays

Stable cell lines can be used in various cell-based assays, such as reporter gene assays, cell proliferation assays, and apoptosis assays, to study cellular processes and signaling pathways.

Cytokine ELISA Strips

Cytokines and growth factors are signaling molecules that have critical roles in many biological processes such as cellular growth, differentiation, gene expression, migration, immunity and inflammation. Disruption in these signals can lead to a variety of diseases, including arthritis, liver disease, inflammatory bowel disease, cardiac-related diseases, and cancers. Signosis provides a number of tools to profile one specific cytokine or growth factor, or to monitor multiple simultaneously. See our ELISA biomarker kit.

Principle

In this assay, the test sample initially reacts with the solid phase capture antibody, resulting in the cytokine being bound to the well. The wells are then washed to remove unbound proteins, and biotin-linked antibodies are added to bind to the cytokines. After washing away the unbound antibodies, Streptavidin-HRP conjugate is added to form a complex with the antibody-bound cytokines.

​For colorimetric kits, the HRP substrate, TMB, is added and forms a blue color when the HRP-linked antibodies are detected. The reaction is then terminated with Stop Solution, which changes the color from blue to yellow. The cytokine concentration in each well is directly proportional to its color intensity and can be quantified by measuring its optical density at 450 nm (OD450) in a microplate reader.

​For chemiluminescence kits, the plate is detected with HRP luminescent substrate. Luminescence is reported as relative light units (RLUs) on a microplate luminometer. The level of expression for each specific cytokine is directly proportional to the luminescent intensity.

Benefits

Multiplex

​One ELISA strip allows the measurement of 8 cytokines.

Flexible

​Comparisons can be made between 2 to 12 samples.

Quantitative Profiling & Analysis

Allows quantitative comparison of expression among samples. Protein standard is available for purchase separately.

Wide Sample Selection

​The assay is suitable for any biological liquid material and cell lysate, such as sera, cerebrospinal fluid (CSF), urine and tissue homogenate.

Rat Cytokine ELISA Strips

Profile 8 different rat cytokines using our ELISA strip kit.

Mouse Cytokine ELISA Strips

Profile 8 different mouse cytokines using our ELISA strip kit

Human Cytokine ELISA Strips

Profile 8 different human cytokines using our ELISA strip kit

Protein Standards

Cytokine ELISA Kits

Cytokines and growth factors are signaling molecules that have critical roles in many biological processes such as cellular growth, differentiation, gene expression, migration, immunity and inflammation. Disruption in these signals can lead to a variety of diseases, including arthritis, liver disease, inflammatory bowel disease, cardiac-related diseases, and cancers. Signosis provides a number of tools to profile one specific cytokine or growth factor, or to monitor multiple simultaneously. See our ELISA biomarker kit.

Principle

In this assay, the test sample initially reacts with the solid phase capture antibody, resulting in the cytokine being bound to the well. The wells are then washed to remove unbound proteins, and biotin-linked antibodies are added to bind to the cytokines. After washing away the unbound antibodies, Streptavidin-HRP conjugate is added to form a complex with the antibody-bound cytokines.

​For colorimetric kits, the HRP substrate, TMB, is added and forms a blue color when the HRP-linked antibodies are detected. The reaction is then terminated with Stop Solution, which changes the color from blue to yellow. The cytokine concentration in each well is directly proportional to its color intensity and can be quantified by measuring its optical density at 450 nm (OD450) in a microplate reader.

​For chemiluminescence kits, the plate is detected with HRP luminescent substrate. Luminescence is reported as relative light units (RLUs) on a microplate luminometer. The level of expression for each specific cytokine is directly proportional to the luminescent intensity.

Colorimetric ELISA Diagram

Benefits

Cost-Effective Analysis

​High Quality with the most competitive price.

Efficient & Flexible

​Multiple samples can be analyzed simultaneously.

Specific & Simple Procedure

A pair of highly selective antibodies against specific cytokine. All in one system with a pre-coated 96-well plate.

Wide Sample Selection

​The assay is suitable for any biological liquid material and cell lysate, such as sera, cerebrospinal fluid (CSF), urine and tissue homogenate.

Rat Cytokine ELISA Kits

Analyze a specific rat cytokine using our ELISA kit

Mouse Cytokine ELISA Kits

Analyze a specific mouse cytokine using our ELISA kit

Human Cytokine ELISA Kits

Analyze a specific human cytokine using our ELISA kit

Associated Kits and Components

Signosis offers an extra supply of reagents alongside associated kits needed for certain products.

Benefits

Cost-Effective Analysis

​High Quality with the most competitive price.

Efficient & Flexible

​Multiple samples can be analyzed simultaneously.

Specific & Simple Procedure

A pair of highly selective antibodies against specific cytokine. All in one system with a pre-coated 96-well plate.

Wide Sample Selection

​The assay is suitable for any biological liquid material and cell lysate, such as sera, cerebrospinal fluid (CSF), urine and tissue homogenate.

Cytokine ELISA Mix and Match

Cytokines and growth factors are signaling molecules that have critical roles in many biological processes such as cellular growth, differentiation, gene expression, migration, immunity and inflammation. Disruption in these signals can lead to a variety of diseases, including arthritis, liver disease, inflammatory bowel disease, cardiac-related diseases, and cancers. Signosis’ Customized Mix and Match ELISA kits can monitor multiple different cytokines of your choice simultaneously. You can choose any cytokine from our wide selection of human, mouse, and rat cytokines.

Cytokine Panel

Principle

In this assay, the test sample initially reacts with the solid phase capture antibody, resulting in the cytokine being bound to the well. The wells are then washed to remove unbound proteins, and biotin-linked antibodies are added to bind to the cytokines. After washing away the unbound antibodies, Streptavidin-HRP conjugate is added to form a complex with the antibody-bound cytokines.

​For colorimetric kits, the HRP substrate, TMB, is added and forms a blue color when the HRP-linked antibodies are detected. The reaction is then terminated with Stop Solution, which changes the color from blue to yellow. The cytokine concentration in each well is directly proportional to its color intensity and can be quantified by measuring its optical density at 450 nm (OD450) in a microplate reader.

Colorimetric ELISA Diagram

Benefits

Multiplex

​One ELISA strip allows the measurement of 8 cytokines.

Flexible

​Comparisons can be made between 2 to 12 samples.

Quantitative Profiling & Analysis

Allows quantitative comparison of expression among samples. Protein standard is available for purchase separately.

Wide Sample Selection

​The assay is suitable for any biological liquid material and cell lysate, such as sera, cerebrospinal fluid (CSF), urine and tissue homogenate.

miRNA Real Time PCR

With the miRNA Real-Time PCR kits, miRNA can be analyzed from total RNA or from cell lysates directly without cDNA conversion. This kit can be used for 12 samples and 50 PCR reactions.

Principle

In the assay, the target miRNA molecule is hybridized with two oligos to form a RNA/DNA duplex. When the sequences are perfectly matched, they are aligned with the miRNA and the joint can be ligated with DNA ligase. A single nucleotide difference among miRNAs will block either the hybridization or the ligation. After the pair of oligos is ligated, the ligated molecules are subjected to real-time PCR analysis.

Benefits

Convenient

All reagents from cell lysis to PCR, are included in one kit

No RNA Preparation

Cell lysates can be used directly in reverse transcription, although total RNA is perfectly fine.

Real-Time

Analysis can be monitored in real time.

miRNA Northern Blot Assays

The assay kits are non-radioactive with all reagents included, and the probes are biotin pre-labeled. Signosis also provides the Highly Sensitive Northern Blot Assay kits, which are 100 times more sensitive than conventional chemiluminescent detection.

Principle

RNA samples are separated through gel electrophoresis and transferred onto a membrane. Expression of a specific miRNA is detected with a biotin-labeled probe. This regular Northern blot assay is suitable to most moderate to high expressors of miRNA. High sensitive Northern blot (NB-1001 and HP-XXXX) is needed to analyze low expressors of miRNA. The biotin-labeled probes contain two moieties – complementary sequence of the miRNA and a tag sequence. The tag sequence is then detected by an amplifier enriched with biotin molecules.

Benefits

No Isotope required

Safe method of detection using chemiluminescence.

No Probe Preparation

Pre-labeled biotin probes included in each kit.

All Components in One Kit

Everything needed to perform the assay is included.

Simple Procedure

No miRNA isolation required, total RNA can be used directly.

miRNA Key Components

Signosis offers an extra supply of reagents needed for certain products.

miRNA Luciferase Reporter Vectors

miRNAs have been shown to regulate many developmental and physiological processes, and the deregulation of miRNAs has been linked to a number of disease processes, including cancer. miRNAs play important roles in fine-tuning gene expression regulators referred to as “dimmer switches” because of their ability to repress gene expression without completely silencing it. The precise monitoring of miRNA expression particularly in cell-based assay, can help to reveal the potential miRNA impacts on cancer etiology, differential stages and therapy development. Signosis developed the miRNA reporter vectors. Each reporter vector was constructed by inserting miRNA exactly complementary sequence (~20nt) as corresponding miRNA binding sites on the downstream of luciferase reporter gene. When miRNA in cells binds to the complementary site at 3’UTR downstream of reporter luciferase gene, it leads to repression of the expression of luciferase and results in reduction of luciferase enzyme activity. The miRNA expression level quantitatively corresponds with the luciferase activities.

100+ luciferase reporter vectors are available for monitoring different miRNAs in vivo. Signosis also provides custom services for any unlisted miRNA that you might be interested in.

Principle

The specific miRNA complementary sequence has been engineered at 3′ untranslated region of luciferase gene to make a miRNA luciferase reporter vector. When miRNA targets the binding site at 3’UTR downstream of reporter luciferase gene, it leads to repression of the expression of luciferase and results in reduction of luciferase enzyme activity. Through comparison of luciferase activity in two samples, up- or down-regulated miRNA can be quantitatively measured.

Benefits

Functional Assay

Monitor miRNA expression in cells

Quantitative Analysis

Expression and activation of miRNAs are measured through suppression of firefly luciferase gene expression.

miRNA Arrays

The miRNA Arrays are used for simultaneously profiling up to 132 miRNAs.

Principle

miRNA is different from large messenger RNA in three aspects; (1) miRNAs are small size molecules with quite a big difference in abundance, (2) mature miRNAs co-exist with their precursor pre-miRNA and pri-miRNA, which only differ in length, and (3) many miRNAs are very closely related in sequences, such as isoforms, only differing by one or a few nucleotides. Therefore, the conventional mircoarray technologies cannot directly be applied to analyzing these molecules. A number of miRNA microarray products are commercially available, but they are either tedious in requiring pre-isolation of microRNA, lack the discriminative power to differentiate between isoforms, or are not sensitive enough to monitor low abundant miRNAs.

In our array assay, each miRNA molecule is targeted by two oligos that hybridize with the target miRNA to form a RNA/DNA duplex. When the sequences are perfectly matched, the oligos are aligned with the miRNA and the joint can be ligated by DNA ligase (figure 1). A single nucleotide difference among miRNAs will block either the hybridization or the ligation; Thus, miRNA isoforms can be differentiated. Due to the small size of miRNA, the hybrid might not be stable; Therefore, we introduce the stacking sequences. By extending these two oligos along with their complementary oligos, the stability is increased. Once the pair of oligos is ligated, the ligated molecules are subjected to linear amplification via T7 transcription into RNA in the presence of biotin-UTP, which are used as probes for array hybridization. To differentiate each isoform, we assigned unique tag sequences to the ligation oligos, so that single nucleotide differences are converted into unique tag sequences. In this way, each isoform can be easily distinguished by array hybridization.

The assay procedure includes three steps: (1) mix the miRNA with provided oligos to form miRNA/oligo hybrids; (2) select the hybrids and remove free oligos, and ligate miRNA-directed pairing of oligos to become a single DNA; and (3) amplify the ligated DNA with T7 transcription.

Benefits

Highly discriminative power

​It is able to differentiate all miRNA isoforms. All let7 isoforms can be clearly distinguished in the assay.

Linear amplification

Captured miRNAs are subjected to T7 amplification without introduction of bias in PCR.

Simple procedure

​The assay is simple and straightforward.

miRNA Plate Assays

With the Plate Assay kits, the conversion of miRNA into cDNA is not needed. It is 100 times more sensitive than Northern Blot Assay, and the discrimination power of distinguishing the isomers of miRNAs is also higher.

Principle

In the proprietary miRNA plate assay, one miRNA molecule is flanked by a capture oligo and a biotinated detection oligo through two bridges oligos. One of the bridge oligos is partially hybridized with the miRNA molecule and the capture oligo and another with the miRNA and the detection oligo. The hybrid is immobilized onto plate through hybridization with an immobilized oligo and detected by a streptavidin-HRP conjugate and chemiluminecscent substrate. This hybrid structure is sensitive to the sequence of the miRNA molecule. One nucleotide difference will prevent the formation of the hybrid and therefore miRNA isoform can be differentiated, which normally is hard to tackle with Northern blot. In addition, the sensitivity of the assay is higher than miRNA Northern blot assay.

Benefits

Simple

​Do miRNA detection as doing ELISA. It does not need enzymatic conversion from miRNA into cDNA. The entire assay procedure is “incubation and wash”.

Higher Sensitivity & Increased Discrimination Power

The detection is approximately 100 times more sensitive than miRNA Northern blot. In addition, it has higher discrimination power than miRNA Northern blot in differentiating miRNA isoforms.