Primer-only premixed assays for analyzing gene expression using intercalating dyes
PrimeTime qPCR Primer Assays provide a primer pair designed for real-time PCR using intercalating dyes, such as SYBR® Green (Molecular Probes) or EvaGreen® (Biotium) dyes. Predesigned assays for human, mouse, or rat are designed with advanced bioinformatic and thermodynamic sequence analytics and for easy selection. Custom assays may be created for any sequence from any species using the PrimerQuest® tool.
Available in standard size, premixed, and shipped dry.
1 Available for human, mouse, and rat targets (for assay configuration, choose intercalating dyes, primers only)
2 Choose design: qPCR–2 primers–intercalating dyes
PrimeTime qPCR Primer Assays are ideal for use with SYBR Green, EvaGreen, and other intercalating dyes, where no probe is needed. PrimeTime qPCR Primer Assays are available in standard size, premixed, normalized to 5 nmol per primer, and shipped dried down. Primer sequences are provided upon order, and the assays ship in 2–3 business days.
Predesigned assays are designed using a proprietary algorithm: in addition to optimized oligo Tm (base composition, oligo length, etc.), the bioinformatic calculations account for factors, such as SNPs (based on current NCBI RefSeq releases), cross-react searches to avoid off-target amplification, recognition of splice variants, and secondary structure predictions.
We provide primer sequences with each order to assist with best practices in research reporting and reproducibility. Specifically, sequence transparency
Figure 1. PrimeTime Assays yield the same high efficiency whether used with intercalating dyes or probe. Amplification of 5 sequential 4-fold dilutions of cDNA using PrimeTime qPCR Primer Assays (with SYBR Green dye) or the PrimeTime qPCR 5′ Nuclease Assay (with dual-labeled probe) targeting human 3-oxoacid CoA transferase 1 (OXCT1) (NM_000436).
Figure 2. PrimeTime qPCR Primer Assays have average reaction efficiency >90%. Sixty randomly selected PrimeTime qPCR Primer Assays and 15 PrimeTime qPCR Primer Assays for endogenous control genes used with Brilliant III Ultra Fast SYBR Green qPCR Master Mix (Agilent) were analyzed over 5 sequential 4-fold dilutions (50–0.195 ng/reaction) of cDNA prepared from Universal Human Reference RNA (Agilent). Reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher) using PCR cycling conditions: 3 min 95°C; 45 x (5 sec 95°C, 15 sec 60°C). Average reaction efficiencies for the assays tested exceeded 98%.