Primetime® qPCR Assays, Primers, and Probes

IDT has developed a new internal ZEN quencher which enables the production of Double-Quenched Probes with less background and increased signal! Double-Quenched Probes contain a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal ZEN quencher. While traditional probes have around 20-30 bases between the fluorophore and the quencher, the internal ZEN quencher decreases that length to only 9 bases. This shortened distance, particularly when combined with the traditional 3’ end quencher, leads to a much more thorough quenching with much less background and enables the use of much longer probes for designing in AT-rich target regions. In addition to the significantly decreased background, the Double-Quenched Probes also have consistently reduced Cq values and improved precision when compared to traditional probes. Use of the Double-Quenched Probes can allow users to experience both increased sensitivity and precision in their qPCR experiments.

For more information download the ZEN Double-Quenched Probe Overview.

IDT and Biogazelle are working together to provide users of PrimeTime® qPCR products a solution for qPCR data analysis. Receive 35% off the list price of a premium qbase^PLUS license from Biogazelle.

Biogazelle is the real-time PCR data analysis company, built upon extensive experience in real-time PCR experiment design, assay development, and data-analysis. Groundbreaking publications on normalization of gene expression and data analysis by Biogazelle founders, Jo Vandesompele and Jan Hellemans, have been cited more than 3000 times.

qbase^PLUS software from Biogazelle is a qPCR analysis package that allows direct import of data from qPCR instruments from a variety of manufacturers. The software is based on proven geNorm and qBase technology and enhanced with proprietary algorithms and time-saving features. These algorithms remove data errors, normalize data to one or more reference genes, and correct inter-run variation using inter-run calibrators. qbase^PLUS software offers statistical tools for qPCR data analysis and graphical presentation tools for viewing analyzed data. It is the most powerful, flexible, and user-friendly real-time PCR data analysis software available. qbase^PLUS is compatible with Windows, Mac, and Linux operating systems and meets MIQE guidelines.

For assay re-ordering, it is critical that manufacturing be reproducible from lot to lot. IDT tested five genes from two lots of PrimeTime^® qPCR Assays with three replicates each. The lots were highly reproducible with very little CQ variation.

Fig. 3A

Aside from lot to lot variation, it is critical that the performance of the assay remain consistent across scales. IDT tested the Mini, Standard and XL PrimeTime^® qPCR Assays and found reproducibility and precision across all three scales. This attribute simplifies transition from discovery or validation applications to screening applications.

Fig. 3B

To demonstrate the performance of different Dye-Quencher combinations, IDT tested a dilution series and found robustness in PCR efficiency and R2 values across all Dye-Quencher combinations available. 

PrimeTime^® Assays Excel with Fast-Cycling Protocols

• No sacrifice in efficiency. All PrimeTime Assays maintained efficiencies between 90–100%. 2 of the 6 Competitor A assays had efficiency values < 90%.
• Greater sensitivity. PrimeTime qPCR Assays had lower C_q values compared to matched, inventoried assays from Competitor A by over 0.5 on average and ΔRn values that were almost 20% higher.

Increased Sensitivity

25 assays from Competitor A were compared to an equal number of IDT PrimeTime^® qPCR Assays. The Competitor A assays consisted of 15 inventoried assays and 10 made-to-order assays. To ensure an accurate comparison was made, the PrimeTime Assays and Competitor A assays were selected to span the same exon boundary of each gene. The reactions were run with the Applied Biosystems Gene Expression Master Mix and identical thresholds were set for all runs (Figures 6 and 7).

Higher qPCR Efficiency 

qPCR efficiency was measured using a comparison of 25 Competitor A and IDT assays for sensitivity (Figure 8). Again, the PrimeTime^® qPCR Assays have a higher average qPCR efficiency than Competitor A assays. In addition, the overall distribution of qPCR efficiency was narrower and higher than that for Competitor A assays.