Ultrasensitive Reporter Gene Assays

NovaBright reporter gene assays offer chemiluminescent measurement of reporter gene activity that is more sensitive than colorimetric or fluorescent detection. NovaBright kits are available for the most common reporter genes, and are optimized to yield consistent results with high sensitivity over a wide dynamic range spanning femtogram to nanogram amounts of reporter.

  • High Sensitivity
  • Wide Dynamic Range
  • Three Easy-to-Use Formats
   

View the NovaBright™ Reporter Gene Assay Selection Guide

Reporter Gene Assays

Studying the Regulation of Gene Expression
Reporter gene assays are invaluable for studying the regulation of gene expression by cis-acting factors (gene regulatory elements) or trans-acting factors (transcription factors or exogenous regulators). In these assays, the reporter gene acts as a surrogate for the coding region of the gene under study.

The reporter gene construct contains one or more gene regulatory elements being analyzed, the structural sequence of the reporter gene, and the sequences required for the formation of functional mRNA. Upon introduction of the reporter construct into cells, expression levels of the reporter gene are monitored through a direct assay of the reporter protein’s enzymatic activity.

Ultra Sensitivity
The sensitivity of each reporter gene assay is a function of several factors including detection method, reporter mRNA and protein turnover, and endogenous (background) levels of the reporter activity. Both protein turnover and levels of endogenous background vary with each reporter protein and the cell line used. Commonly used detection techniques utilize isotopic, colorimetric, fluorometric, or luminescent enzyme substrates and immunoassay-based procedures with isotopic, colorimetric, or chemiluminescent endpoints.

Common Reporter Genes

Beta-galactosidase
Beta-Galactosidase is traditionally detected with the colorimetric substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) [1], and is often used in conjunction with other reporter genes to normalize transfection efficiency. As indicated in the table [web designer link to table], this colorimetric assay is less sensitive compared to many other reporter gene assays. With NovaBright 1,2-dioxetane chemiluminescent substrates for beta-galactosidase, the sensitivity is increased dramatically [2,3].

See More Common Reporter Genes >


Secreted Placental Alkaline Phosphatase
Secreted placental alkaline phosphatase (SEAP) is secreted by cells directly into the culture medium, and can be assayed simply by taking samples of cell culture medium. Secreted reporter proteins enable the nondestructive assay of cell culture medium, preserving cells for additional assays and enabling time-course monitoring of gene expression. SEAP is detected with both colorimetric and chemiluminescent substrates. NovaBright™ chemiluminescent SEAP reporter gene assays exhibit remarkable sensitivity and ease of use.

Luciferase
Luciferase has become increasingly popular as a reporter gene, especially for cotransfection experiments where it is important to normalize transfection efficiency. The NovaBright β-galactosidase and firefly luciferase dual enzyme reporter gene assay offers high sensitivity and a simple assay procedure, and enables the user to perform both measurements from a single aliquot of cell extract in the same reaction well or tube, which minimizes experimental error.

Featured data & citations

Reporter Gene
Detection MethodDetection LimitAdvantagesDisadvantages
Chloramphenicol acetyltransferase (CAT)Isotopic,
ELISA		5 x 107 molecules,
1 x 109 molecules		Widely used,
No radioactivity		Radioactive,
High cost,
Low dynamic range,
Labor intensive,
High cost per assay
Beta-galactosidaseONPG (color),
MUG (Fluorescence),
NovaBright™ beta-galactosidase system,
NovaBright™ SEAP system		3 x 108 molecules,
6 x 105 molecules,
3 x 103 molecules		High sensitivity,
Wide dynamic range,
Simplicity		Poor sensitivity,
Autofluorescence
Human growth hormoneRadioimmunoassay3 x 108 moleculesSecreted into mediaRadioactivity,
High cost per assay,
Low sensitivity
LuciferaseNovaBright™ dual beta-galactosidase and firefly luciferase system103–104 moleculesAssay simplicity,
Wide dynamic range		Protein instability
Secreted placental alkaline phosphatasepNPP (color),
NovaBright™ SEAP systems		1 x 108 molecules,
3 x 104 molecules		Secreted into media,
High Sensitivity,
Wide dynamic range		Poor sensitivity

NovaBright citations

Review our comprehensive list of publications that used NovaBright™ reporter gene assay products.

1. Alexander L, Illyinskii PO, Lang SM et al. (2003) Determinants of increased replicative capacity of serially passaged Simian Immunodeficiency Virus with nef deleted in Rhesus monkeys. J Virol 77:6823–6835.
2. Berger J, Hauber J, Hauber R et al. (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66:1–10.
3. Bronstein I, Fortin JJ, Voyta JC et al. (1994a) Chemiluminescent reporter gene assays: Sensitive detection of the GUS and SEAP gene products. Biotechniques 17:172–178.
4. Brown MA, Zhao Q, Baker KA et al. (2005) Crystal structure of BMP-9 and functional interactions with pro-region and receptors. J Biol Chem 280:25111–25118.
5. Cao HB, Wang A, Martin B et al. (2005) Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference. Cell Res 15:111–119.
6. Cullen B, Malim M (1992) Secreted placental alkaline phosphatase as a eukaryotic reporter gene. MethodsEnzymol 216:362–368.
7. Duboise M, Guo J, Czajak S et al. (1998) A role for Herpesvirus Saimiri orf14 in transformation and persistent infection. J Virol 72:6770–6776.
8. Hobbs WE, Brough DE, Kovesdi I et al. (2001) Efficient activation of viral genomes by levels of Herpes Simplex Virus ICP0 insufficient to affect cellular gene expression or cell survival. J Virol 75:3391–3403.
9. Hwang DR, Tsai YC, Lee JC et al. (2004) Inhibition of Hepatitis C virus replication by arsenic trioxide. Antimicrob Agents Chemother 48:2876–2882.
10. Johnson WE, Morgan J, Reitter J et al. (2002) A replication-competent, neutralization-sensitive variant of Simian Immunodeficiency Virus lacking 100 amino acids of envelope. J Virol 76:2075–2086.
11. Kagan JC, Stein MP, Pypaert M et al. (2004) Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle. J Exp Med 199:1201–1211.
12. Khan AS, Smith LC, Abruzzese RV et al. (2003) Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs. DNA Cell Biol 22:807–814.
13. Latta-Mahieu M, Rolland M, Caillet C et al. (2002) Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther 13:1611–1620.
14. Pohlmann S, Krumbiegel M, Kirchhoff F (1999) Coreceptor usage of BOB/GPR15 and Bonzo/STRL33 by primary isolates of human immunodeficiency virus type 1. J Gen Virol 80:1241–1251.
15. Poser S, ImpeyS, Xia Z et al. (2003) Brain-derived neurotrophic factor protection of cortical neurons from serum withdrawal-induced apoptosis is inhibited by cAMP. J Neurosci 23:4420–4427.
16. O’Connor KL, Culp LA (1994) Quantitation of two histochemical markers in the same extract using chemiluminescent substrates. BioTechniques 17:502–509.
17. Riera M, Chillon M, Aran JM et al. (2004) Intramuscular SP1017-formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues. J Gene Med 6:111–118.
18. Rubenstrunk A, Orsini C, Mahfoudi A et al. (2003) Transcriptional activation of the metallothionein I gene by electric pulses in vivo: Basis for the development of a new gene switch system. J Gene Med 5:773–783.
19. Sakai J, Rawson RB, Espenshade PJ et al. (1998) Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. Mol Cell 2:505–514.
20. Zabeau L, Defeau D, Van der Heyden J et al. (2004) Functional analysis of leptin receptor activation using a Janus kinase/signal transducer and activator of transcription complementation assay. Mol Endocrinol
21. Zhang J, Ou J, Bashmakov Y et al. (2001) Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes. Proc Natl Acad Sci U S A
22. Zhao H, Peeters BPH (2003) Recombinant Newcastle Disease Virus as a viral vector: Effect of genomic location of foreign gene on gene expression and virus replication. J Gen Virol 84:781–788.

Comparison of reporter gene assays
Reporter Gene
Detection MethodDetection LimitAdvantagesDisadvantages
Chloramphenicol acetyltransferase (CAT)Isotopic,
ELISA		5 x 107 molecules,
1 x 109 molecules		Widely used,
No radioactivity		Radioactive,
High cost,
Low dynamic range,
Labor intensive,
High cost per assay
Beta-galactosidaseONPG (color),
MUG (Fluorescence),
NovaBright™ beta-galactosidase system,
NovaBright™ SEAP system		3 x 108 molecules,
6 x 105 molecules,
3 x 103 molecules		High sensitivity,
Wide dynamic range,
Simplicity		Poor sensitivity,
Autofluorescence
Human growth hormoneRadioimmunoassay3 x 108 moleculesSecreted into mediaRadioactivity,
High cost per assay,
Low sensitivity
LuciferaseNovaBright™ dual beta-galactosidase and firefly luciferase system103–104 moleculesAssay simplicity,
Wide dynamic range		Protein instability
Secreted placental alkaline phosphatasepNPP (color),
NovaBright™ SEAP systems		1 x 108 molecules,
3 x 104 molecules		Secreted into media,
High Sensitivity,
Wide dynamic range		Poor sensitivity
NovaBright citations

NovaBright citations

Review our comprehensive list of publications that used NovaBright™ reporter gene assay products.

1. Alexander L, Illyinskii PO, Lang SM et al. (2003) Determinants of increased replicative capacity of serially passaged Simian Immunodeficiency Virus with nef deleted in Rhesus monkeys. J Virol 77:6823–6835.
2. Berger J, Hauber J, Hauber R et al. (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66:1–10.
3. Bronstein I, Fortin JJ, Voyta JC et al. (1994a) Chemiluminescent reporter gene assays: Sensitive detection of the GUS and SEAP gene products. Biotechniques 17:172–178.
4. Brown MA, Zhao Q, Baker KA et al. (2005) Crystal structure of BMP-9 and functional interactions with pro-region and receptors. J Biol Chem 280:25111–25118.
5. Cao HB, Wang A, Martin B et al. (2005) Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference. Cell Res 15:111–119.
6. Cullen B, Malim M (1992) Secreted placental alkaline phosphatase as a eukaryotic reporter gene. MethodsEnzymol 216:362–368.
7. Duboise M, Guo J, Czajak S et al. (1998) A role for Herpesvirus Saimiri orf14 in transformation and persistent infection. J Virol 72:6770–6776.
8. Hobbs WE, Brough DE, Kovesdi I et al. (2001) Efficient activation of viral genomes by levels of Herpes Simplex Virus ICP0 insufficient to affect cellular gene expression or cell survival. J Virol 75:3391–3403.
9. Hwang DR, Tsai YC, Lee JC et al. (2004) Inhibition of Hepatitis C virus replication by arsenic trioxide. Antimicrob Agents Chemother 48:2876–2882.
10. Johnson WE, Morgan J, Reitter J et al. (2002) A replication-competent, neutralization-sensitive variant of Simian Immunodeficiency Virus lacking 100 amino acids of envelope. J Virol 76:2075–2086.
11. Kagan JC, Stein MP, Pypaert M et al. (2004) Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle. J Exp Med 199:1201–1211.
12. Khan AS, Smith LC, Abruzzese RV et al. (2003) Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs. DNA Cell Biol 22:807–814.
13. Latta-Mahieu M, Rolland M, Caillet C et al. (2002) Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther 13:1611–1620.
14. Pohlmann S, Krumbiegel M, Kirchhoff F (1999) Coreceptor usage of BOB/GPR15 and Bonzo/STRL33 by primary isolates of human immunodeficiency virus type 1. J Gen Virol 80:1241–1251.
15. Poser S, ImpeyS, Xia Z et al. (2003) Brain-derived neurotrophic factor protection of cortical neurons from serum withdrawal-induced apoptosis is inhibited by cAMP. J Neurosci 23:4420–4427.
16. O’Connor KL, Culp LA (1994) Quantitation of two histochemical markers in the same extract using chemiluminescent substrates. BioTechniques 17:502–509.
17. Riera M, Chillon M, Aran JM et al. (2004) Intramuscular SP1017-formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues. J Gene Med 6:111–118.
18. Rubenstrunk A, Orsini C, Mahfoudi A et al. (2003) Transcriptional activation of the metallothionein I gene by electric pulses in vivo: Basis for the development of a new gene switch system. J Gene Med 5:773–783.
19. Sakai J, Rawson RB, Espenshade PJ et al. (1998) Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. Mol Cell 2:505–514.
20. Zabeau L, Defeau D, Van der Heyden J et al. (2004) Functional analysis of leptin receptor activation using a Janus kinase/signal transducer and activator of transcription complementation assay. Mol Endocrinol
21. Zhang J, Ou J, Bashmakov Y et al. (2001) Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes. Proc Natl Acad Sci U S A
22. Zhao H, Peeters BPH (2003) Recombinant Newcastle Disease Virus as a viral vector: Effect of genomic location of foreign gene on gene expression and virus replication. J Gen Virol 84:781–788.

Comparison of Reporter Gene Assays

Reporter Gene
Detection MethodDetection LimitAdvantagesDisadvantages
Chloramphenicol acetyltransferase (CAT)Isotopic,
ELISA		5 x 107 molecules,
1 x 109 molecules		Widely used,
No radioactivity		Radioactive,
High cost,
Low dynamic range,
Labor intensive,
High cost per assay
Beta-galactosidaseONPG (color),
MUG (Fluorescence),
NovaBright™ beta-galactosidase system,
NovaBright™ SEAP system		3 x 108 molecules,
6 x 105 molecules,
3 x 103 molecules		High sensitivity,
Wide dynamic range,
Simplicity		Poor sensitivity,
Autofluorescence
Human growth hormoneRadioimmunoassay3 x 108 moleculesSecreted into mediaRadioactivity,
High cost per assay,
Low sensitivity
LuciferaseNovaBright™ dual beta-galactosidase and firefly luciferase system103–104 moleculesAssay simplicity,
Wide dynamic range		Protein instability
Secreted placental alkaline phosphatasepNPP (color),
NovaBright™ SEAP systems		1 x 108 molecules,
3 x 104 molecules		Secreted into media,
High Sensitivity,
Wide dynamic range		Poor sensitivity

References

  1. Alam J, Cook JL (1990) Reporter genes: application to the study of mammalian gene transcription. Anal Biochem 188:245–254.
  2. Bronstein I, Martin CS, Fortin JJ, Olesen CE, Voyta JC (1996) Chemiluminescence: Sensitive detection technology for reporter gene assays. Clin Chem 42:1542–1546.
  3. Bronstein I, Fortin J, Stanley PE, Stewart GS, Kricka LJ (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal Biochem 219:169–181.

Resources

For Research Use Only. Not for use in diagnostic procedures.