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Selecting qPCR Assays in Plates for High Throughput and Large-Scale Studies

The PrimeTime® Plates Design Tool

The PrimeTime® Plates Design Tool is a state-of-the-art online tool for easy selection and ordering of PrimeTime qPCR Assays for gene expression analysis in human, mouse, and rat. The design tool, which is accessible from both the main Ordering page and the Gene Expression products page (Figure 1), has 2 interfaces for ordering either 5’ nuclease (probe-based) qPCR assays or primer-based qPCR assays for use with intercalating dyes.



         A. Access the PrimeTime® Plates Ordering Tool from the Main Ordering Page.



        B. Access the Gene Expression Products Page.

Figure 1. Routes to Easy qPCR Plate Design.

Phase 1: Selection from PrimeTime Predesigned Assay Library

  1. Choose from our predesigned collection of qPCR assays covering the human, mouse, and rat transcriptomes (Figure 2). These predesigned assays use up-to-date sequence information from the NCBI RefSeq database and predict Tm and secondary structures under standard qPCR conditions to prevent off-target amplification and avoid SNPs.
  2. Choose assays suitable for your needs based on splice variants or exon locations.
  3.  Select desired assays by checking the appropriate boxes and click Add To Plate. There may be several pages of assays; however, only assays from the page displayed will be added to the plate. To select assays from subsequent pages, you must display them. A minimum order of only 24 assays is required. If more than 96 assays are selected, additional plates will be added automatically.
  4. The Pathway Gene List link provides the added benefit of viewing and copying commonly described pathways and gene lists.
  5. After selecting the desired assays from the library, you can begin the plate design process.

            A. Find Predesigned Assays For Human, Mouse, or Rat Sequences.



             B. Select Assays Based on Species and Gene Symbol, RefSeq ID, or Assay ID.


Figure 2. Select PrimeTime® Predesigned qPCR Assays from the Library.


Phase 2: User-Controlled Arrangement of Assays

  1. Select the appropriate scale. Material for multiple reactions is provided in each well. Choose the scale based on the number of samples you plan to analyze.
  2. Select the primer-to-probe ratio. Note that the PrimeTime Plates Design Tool is the only tool that lets researchers define custom primer-to-probe ratios, and these can be varied across the plate.
  3. To rearrange assays or create duplicate wells, select a well (or range of wells) and use Ctrl + X, Ctrl + C, and Ctrl + V for cut, copy, and paste, re­spectively (for a Mac computer, use the Command key and the appropriate letter key), as you would for Microsoft Excel.
  4. For probe-based assays, you can also specify and change dye selections. Each well can have only one dye–quencher combination; however, you may select different dye–quencher combinations across the plate wells (Figure 3).

 

Figure 3. Multiple Dye–Quencher Combinations Can Be Selected in Each Plate.

For intercalating dye primer sets (e.g., for SYBR® Green assays), all of the features described above are replicat­ed with the exception of dye selection and adjustment of primer:probe ratio as no probe is required. Addition­ally, primer assays are available only at standard scale (Figure 4).

Figure 4. User Interface for Designing PrimeTime® Plates for Intercalating Dye Assays.

The IDT PrimeTime Plates Design Tool is unique in its sophistication, flexibility, and ease of use—researchers appreciate that they are not limited only to selecting predesigned assays from the library. Custom assay de­signs can be combined with predesigned assays in the same plate (Figure 5). This ability to mix and match in a plate design tool is unique to IDT and gives researchers the utmost flexibility and choice to design plates that best suit their needs.

 Figure 5. Flexible Plate Design for Combining Predesigned and Custom Assays.

Author: Nicola Brookman-Amissah, PhD, is a Scientific Writer at IDT.