Use splice junctions to your advantage in qPCR

Quantitative, real-time PCR (qPCR; Figure 1) has become the gold standard for gene expression analysis. This method is fast, accurate, and sensitive, and requires less sample input than for northern blots, ribonuclease protection assays, semi-quantitative RT-PCR, and competitive RT-PCR.

Figure 1. A typical qPCR workflow. For accurate gene expression results, researchers typically design across exon-exon junctions to avoid genomic DNA (gDNA) amplification and detection. Using RNA isolation protocols that minimize DNA contamination or including a DNase treatment step can also help avoid gDNA amplification.

How qPCR assays span splice junctions

qPCR assays designed to span exon-exon junctions can be used to distinguish and quantify splice variants, detect all splice variants, or even detect species-specific gene expression. Depending on the structure of your gene of interest, there is more than one way to design primers that span exon-exon boundaries. For example, one of the primers can be designed with one end complementary to the 3′ end of one exon and the other end complementary to the 5′ end of the next downstream exon (Figure 2A), so that amplification will only occur if this primer binds to cDNA from a spliced mRNA transcript. Alternatively, the forward and reverse primers can be designed to hybridize to different exons that are separated by a large intron or large intron-exon region (Figure 2B and 2C). Under typical PCR cycling conditions, amplification of the smaller cDNA-based amplicon will be favored over the larger gDNA-based amplicon.

Figure 2. qPCR primer designs that span splice junctions. (A) To prevent amplification of incompletely spliced transcripts, one of the primers is designed to overlap an exon-exon junction.(B) Primers can also be designed to hybridize with sequences in consecutive exons. (C) For splice variant detection, primers may span a region that contains more than one small intron and exon, for a resulting amplicon of 70–200 bp.

There are a few situations where you might need to design a qPCR assay with a probe that spans the exon-exon junction (e.g., for expression analysis of some alternatively spliced genes). However, if the primers amplify gDNA, PCR efficiency of the desired amplicon could be affected, since gDNA amplification will not be detected by a probe using this design strategy.

Tip #1: For human, mouse, and rat genes, select from PrimeTime Predesigned qPCR Assays

Up-to-date, reliable predesigned assays

Combining expertise in programming, bioinformatics, molecular biology, and nucleic acid thermodynamics, IDT scientists have developed a robust design algorithm to produce a collection of PrimeTime Predesigned qPCR Assays for most human, rat, and mouse genes. This design process uses the cleanest possible target sequence information (including up-to-date SNP and intron-exon junction locations), and stresses accurate T_m prediction and protection against off-target amplification. The algorithm includes additional secondary design considerations and scoring criteria, such as analysis of polynucleotide runs, short sequence repeats, and hetero- and homo-dimer folding energies.

Using PrimeTime Predesigned Assays

If you are studying human, mouse, or rat genes, the predesigned qPCR assays, which are available with or without probes, allow you to choose from multiple assays that target different locations within a single gene. From the Predesigned qPCR Assays ordering page, you can view the assays in 4 short steps:

1. Enter the Gene Symbol or RefSeq accession number.

Note: If you are reordering an assay, you only need to enter the IDT Assay ID as the search criterion. Assay IDs can be found on your tube labels, order history, and specification sheets. (Representative Assay ID: Hs.PT.58.19228441)

2. Select the species (Human, Mouse, or Rat).
3. Select the desired Assay Configuration (5′ nuclease assay with primers and probe, or intercalating dye assay with only primers).
4. Click Search (see Figure 3 for representative results).

Figure 3. Where to find assay details to help you choose the best predesigned PrimeTime qPCR Assays for your experiments. This image shows representative results with boxes highlighting the columns that will help you choose the best assays for your gene expression experiments, whether your main purpose is avoiding gDNA amplification or quantification of splice variants. In brief, some Assay ID numbers include “.g” or “.gs” suffixes, which indicate that the assay may detect genomic DNA (blue box). The Detects All Variants column indicates whether or not all splice variants are detected by this assay (orange box), while the number in parentheses in the RefSeq # column shows the number of variants detected by the assay. Exon Location provides exon numbers that the assay spans (green box). Amplicons from assays with multiple exons will span the junction(s) between the exons.

Choosing assays that span exon-exon junctions

The results section of the PrimeTime Predesigned qPCR Assay ordering tool provides a wealth of information, some of which will not be covered here, but all details are available online by placing your cursor over, or clicking on, the info buttons. In Figure 3, boxes highlight the results columns that are useful for choosing assay(s) that avoid genomic DNA amplification or that can distinguish cDNAs reverse transcribed from alternatively spliced mRNAs. Here are our recommendations:

Tip #2: When your samples are not from human, mouse, or rat, use the PrimerQuest^® Tool to create custom PrimeTime qPCR Assays

Selecting custom assays

The PrimerQuest Tool is the program of choice for designing qPCR primers/probes, sequencing oligonucleotides, and custom primers. Because of its utility, it is one of the most highly accessed pages on the IDT website. Briefly, the four main steps for using the PrimerQuest Tool include the following:

1. Enter sequence
2. Choose design
3. Customize your design parameters
4. Select and order assays

The PrimerQuest Tool provides live designs that, by default, do not take exon junction locations into consideration, so that assay results can be returned faster. The following tip focuses on Step 3 above—how to adjust the custom design parameters to create primers that span exon-exon junctions.

Finding the appropriate intron-exon boundaries for your gene

Find your sequence in the NCBI nucleotide database using the gene name or GenBank accession number. If you start with the gene name, narrow your search results to your species of interest and choose your sequence. Scroll down to the "Features" section of your sequence results to obtain the exon boundary numbers (for some genes, it may be important to understand transcript variants, exon organization, and SNP locations).

Customizing your qPCR assays

1. Use your GenBank accession number to download your sequence into the PrimerQuest Tool, and click on Show Custom Design Parameters in the Choose Your Design section.
2. Select qPCR (2 Primers + Probe) or qPCR Intercalating Dyes (Primers only) in the box on the right, and scroll down to the Custom Target Region
3. To design one of your primers (or probe) to span an exon-exon junction (Figure 2A), simply enter the nucleotide number(s) of the 3′ end of the exon(s) into the Overlap Junction List (Figure 4). The results page includes the numbers of the starting and ending nucleotides for each primer and probe, so you will be able to tell which oligo overlaps the exon-exon junction.

Figure 3. Adjust settings in Design Across Junctions (blue box) to design assays with primers that span an exon-exon junction. In the Regions Considered schematic, the exon boundaries will be marked in purple under your target sequence.
4. To have your primers hybridize to different exons (Figures 2B and 2C), enter the range(s) of nucleotide numbers ±10 from your exon boundaries in the Target Region List under Primer Focus Region.

Figure 4. Adjust settings in Target Region List (blue box) to design assays with primers that hybridize to different exons. In the Regions Considered schematic, the target region(s) will be marked in light blue under your target sequence.

5. If no assays are found, use the notes at the bottom of the results page to adjust parameters in a new assay search (click on adjust parameters on the results page).

   For example, the results shown in Figure 6 indicate that >375 possible designs for each primer and probe were evaluated (top: Possible designs), and at least 20 met all design requirements for each primer and probe (bottom: Number met design parameters). However, all 1020 possible assay designs produce amplicon sizes outside the specified range (last column: Pair). In this scenario, consider adjusting the boundaries of the excluded region. As further refinements, you might consider adjusting the range of %GC content, the overlap on the 3′ and 5′ end of the primer, and/or the maximum or minimum acceptable T_m.

Figure 6. Representative results from a customized search that failed to produce acceptable qPCR assay. The notes at the bottom (blue box) can be used to adjust design parameters in a follow up assay search.