Real-time, or quantitative, PCR (qPCR) is a powerful and common technique for accurate analysis of gene expression. Our dye-based qPCR kits utilize our proprietary TB Green intercalating dye. This versatile dye can be used with standard methods and equipment with no need for protocol modifications. Green intercalating dye-based qPCR works by the dye binding to double-stranded DNA, which is produced during each PCR cycle. When deciding whether to do probe-based qPCR or intercalating dye-based qPCR, it is important to consider the advantages and limitations of each.

In general, dye-based qPCR is best suited for experiments that require quick and straightforward testing of large numbers of samples for gene expression. This method is comparatively inexpensive as it uses standard, unlabeled oligonucleotides for amplification. This makes it an economical, sensitive option that requires comparatively little optimization and is highly compatible with high-throughput screens and other large-scale studies.

However, dye-based qPCR has some limitations. The green dye intercalates into all double-stranded DNA during the PCR steps, causing nonspecific PCR products to fluoresce. While the proportion of specific detection can be assayed by looking for a single, sharp peak in a melt curve analysis, this requires extra hands-on time and may require additional optimization to eliminate nonspecific products. Finally, predesigned dye-based primer sets are more difficult to find than probes, necessitating a do-it-yourself target validation approach.

We offer multiple formats of dye-based qPCR mixes to meet your experimental needs and give you flexibility in your experimental approaches.

Fluorescent detection using intercalating dyes

This method uses a DNA intercalator (e.g., TB Green) that emits fluorescence when bound to double-stranded DNA. Monitoring fluorescence allows for quantification of amplification products (Figure 1). Following amplification, performing a melt curve analysis provides information on the specificity of your PCR products. To maximize specificity and sensitivity, our kits use Takara Ex Taq DNA Polymerase Hot-Start Version, a hot-start PCR enzyme that minimizes nonspecific amplification that may arise from mispriming or primer-dimer formation during reaction mixture preparation and pre-cycling steps.

Figure 1. Schematic of intercalating-dye-based qPCR.

We have developed multiple different kits and mixes that use TB Green to support your experiments.

• Versatile and efficient master mix that works with most templates—TB Green Advantage qPCR Premix, Cat. # 639676
• Convenient mix for direct qPCR on crude extracts without purification—Terra qPCR Direct TB Green Premix, Cat. # 638319

We also have a series of kits that utilize the heat-resistant Tli RNase H to remove RNA in RNA/DNA hybrids. This RNase exposes primer binding sites of cDNA, especially from GC-rich regions. Further, if RNA is not fully degraded, it may compete for primers and increase your template complexity. Finally, it allows for extension times to be decreased from 30 seconds to 20 seconds.

• Mix for high specificity and longer amplicons—TB Green Premix Ex Taq (Tli RNase H Plus)
  • For maximum flexibility, we have a master mix that comes with two different tubes of ROX dyes for maximum instrument support: TB Green Premix Ex Taq (Tli RNase H Plus), Cat. # RR420A/B
  • For bulk applications that require maximum throughput, we have a master mix with one ROX dye tube in volumes up to 25 ml: TB Green Premix Ex Taq (Tli RNase H Plus), Cat. # RR420L/W
  • For convenience, we have a complete master mix already containing ROX dye in small and large volume formats up to 25 ml: TB Green Premix Ex Taq (Tli RNase H Plus), Cat. # RR42LR/WR
• Mix for maximum specificity and for GC-rich targets—TB Green Premix Ex Taq II (Tli RNase H Plus)
  • For maximum flexibility, we have a master mix that comes with two different tubes of ROX dyes for maximum instrument support: TB Green Premix Ex Taq II (Tli RNase H Plus), Cat. # RR820A/B
  • For bulk applications that require maximum throughput, we have a master mix with one ROX dye tube in volumes up to 25 ml: TB Green Premix Ex Taq II (Tli RNase H Plus), Cat. # RR820L/W
  • For convenience, we have a complete master mix already containing ROX dye in small and large volume formats up to 25 ml: TB Green Premix Ex Taq II (Tli RNase H Plus), Cat. # RR82LR/WR

Here are a few examples of the work that's been driven by our TB Green-based qPCR kits:

Chen, Q., Gu, Y. & Liu, B. Expression and mechanism of action of the SARI tumor suppressor in prostate cancer. Int. J. Clin. Exp. Pathol. 8, 7953–60 (2015).

Cat. # RR820A was used by researchers to identify SARI, a key regulator of prostate cancer proliferation, in tissues and cells.

Chen, X. et al. The role of miRNAs in drug resistance and prognosis of breast cancer formalin-fixed paraffin embedded tissues. Gene 595, 221–226 (2016).

Researchers tested the effects of differential miRNA expression and drug resistance in 55 breast cancer FFPE tissues. Cat. # 639676 was used to identify key miRNAs that could serve as biomarkers for breast cancer treatment.

He, C. et al. The differential expression and possible function of long noncoding RNAs in liver cells infected by dengue virus. Am. J. Trop. Med. Hyg. 97, 1904–1912 (2017).

Researchers performed RNA-seq to identify candidate lncRNAs implicated in liver injury following dengue virus infection. Cat. # 639676 was used by researchers to confirm differentially expressed lncRNAs and identify novel diagnostic markers for disease.

Li, Y. et al. MAF1 suppresses AKT-mTOR signaling and liver cancer through activation of PTEN transcription. Hepatology 63, 1928–42 (2016).

Cat. # 638319 was used by researchers to directly assess MAF1 expression in primary human hepatocellular carcinoma tumors. These data identified novel tumor-suppression activities with potential prognosis prediction value.

Sato, Y. et al. Anks4b, a novel target of HNF4α protein, interacts with GRP78 protein and regulates endoplasmic reticulum stress-induced apoptosis in pancreatic β-cells. J. Biol. Chem. 287, 23236–45 (2012).

Cat. # RR820A was used by researchers to identify Anks4b, a novel target implicated in β-cell apoptosis.

Sung, H. Y. et al. Amyloid beta-mediated hypomethylation of heme oxygenase 1 correlates with cognitive impairment in Alzheimer’s disease. PLoS One 11, e0153156 (2016).

Cat. # RR420A was used by researchers to quantify expression levels of HMOX1, as well as methylation levels of particular CpG sites, implicated in Alzheimer’s disease.