PCR and qPCR
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Use splice junctions to your advantage in qPCR

Tips for simplifying your assay design process

Quantitative, real-time PCR (qPCR) 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. 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 include a DNase treatment step can also help avoid gDNA amplification (see sidebar, qPCR workflow).

In addition, 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.

qPCR workflow

A typical qPCR experiment for gene expression analysis involves the following steps:


  1. Collect sample

  2. Isolate RNA from sample

    Include DNase treatment of your RNA

  3. Perform reverse transcription (RT)

    Include –RT control to help monitor presence of gDNA

  4. Perform real-time PCR

    Use qPCR assays that include a primer (or probe) spanning a splice junction

  5. Validate and analyze qPCR results


qPCR workflow

How qPCR assays span splice junctions

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 1A), 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 1B and 1C). Under typical PCR cycling conditions, amplification of the smaller cDNA-based amplicon will be favored over the larger gDNA-based amplicon.

qPCR primer designs that span splice junctions

Figure 1. 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.

Additional primer and probe design considerations for qPCR experiments are included in the article, Designing PCR primers and probes [1]. These guidelines are part of the design algorithms used to generate PrimeTime® Predesigned qPCR Assays (Tip #1 below) and used by the PrimerQuest Tool (Tip #2 below) to create custom PrimeTime qPCR Assays.


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

Use 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 Tm 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.

Reduce the amount of time spent troubleshooting and optimizing your qPCR assays by using PrimeTime Predesigned qPCR Assays, which are guaranteed to perform with PCR efficiencies of 90–110% and R2 >0.99 (see www.idtdna.com/primetime for details about our guarantee). Primer and probe sequences are provided upon purchase.

Note: if you want to design probes that span exon-exon junctions, see Tip #2 to learn how to customize your PrimeTime assay.

A. Easily identify PrimeTime Predesigned qPCR Assays for your gene

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 (www.idtdna.com/site/order/qpcr/predesignedassay), 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 2 for representative results).

 

Figure 2. Where to find assay details to help you choose the best predesigned PrimeTime qPCR Assays for your experiments

Figure 2. 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.


B. Choose 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 (black info button) or clicking on (blue info button) the info buttons. In Figure 2, 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:

Header Description Recommendation

Assay ID

Suffixes:

  • .g: The assay may detect genomic DNA.

  • .gs: Assays configured as primers only (intercalating dye-base assays) may detect genomic DNA, while assays configured as primers and probe (5′ nuclease assay) will not.

  • To avoid potential amplification of genomic DNA, do not use an assay with a .g suffix.

  • Additionally, if you are using intercalating dye-based assays, do not use an assay with a .gs suffix.

Detects All Variants

  • Yes: All known splice variants are detected by the assay.

  • No: Only some splice variants are detected by the assay. The number in parentheses in the RefSeq # column shows the number of variants detected.

Selection is based on your research goals.

  • If you do not need to discriminate splicing isoforms, choose assays with a “yes”.

  • If you want to examine specific splice variants, choose assays with a “no”. Then review the Exon Location column to determine which variants are detected.

Exon Location*

  • Single exon number: The primers (and probe) hybridize within a single exon, and will likely amplify genomic DNA (e.g., Figure 2, Assay ID Hs.PT.58.24349547.g).

  • Multiple exon numbers: The assay spans multiple exons, such that the PCR amplicon spans the junction between the exons. If the intervening introns and exons are <500 bp, it is possible that some assays will detect genomic DNA (e.g., Figure 2, Assay ID Hs.PT.58.40761082.g).

Use your knowledge about the structure of your gene combined with information about Assay ID suffixes, Variants, and Exon Location to make your assay selection.

* Exon numbering can be complicated, especially when comparing information from various sources. We describe the IDT exon numbering system in the article, Exon numbering—not as easy as 1, 2, 3 [2].


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

The PrimerQuest Tool (www.idtdna.com/primerquest) is the program of choice for designing qPCR primers/probes, sequencing oligonucleotides, and custom primers, making this tool one of the most highly accessed pages on our website. A detailed description of how to use the PrimerQuest Tool is available in the article, Design efficient PCR and qPCR primers and probes using online tools [3]. 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. This tip focuses on Step 3: How to adjust the Custom design parameters to create primers that span exon-exon junctions.

A. Find the appropriate intron-exon boundaries for your gene

Find your sequence in the NCBI nucleotide database (www.ncbi.nlm.nih.gov) 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 (see short video explanation). For some genes, it also may be important to understand transcript variants, exon organization, and SNP locations.

B. Customize your qPCR assays

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.

  1. Be sure to select qPCR (2 Primers + Probe) or qPCR Intercalating Dyes (Primers only) in the box on the right.

  2. Scroll down to the Custom Target Region.

    • To design one of your primers (or probe) to span an exon-exon junction (Figure 1A), simply enter the nucleotide number(s) of the 3′ end of the exon(s) into the Overlap Junction List (Figure 3). 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 to design assays with primers that span an 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. See short video explanation.


      To have your primers hybridize to different exons (Figures 1B and 1C), 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 to design assays with primers that hybridize to different exons.

      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. See short video explanation.


  3. 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 5 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 Tm.

  4. Figure 5. Representative results from a customized search that fialed to produce acceptable qPCR assays

    Figure 5. 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. See short video explanation.


More complex designs?

The PrimerQuest Tool makes it easy to design basic and highly customized primers and probes for PCR and qPCR. Look for future articles from IDT about experiments requiring more complex assay design considerations, such as multiplexing, SNP, and CNV designs.

Need additional assistance with designing assays? Contact applicationsupport@idtdna.com.


References

  1. Prediger E. (2013) Designing PCR Primers and Probes. [Online] Coralville, Integrated DNA Technologies. Available at www.idtdna.com/pages/decoded/decoded-articles/pcr-qpcr/decoded/2013/10/21/designing-pcr-primers-and-probes. [Accessed 18 Jun, 2016].

  2. Downey N. (2013) Exon numbering—not as easy as 1, 2, 3… [Online] Coralville, Integrated DNA Technologies. Available at www.idtdna.com/pages/decoded/decoded-articles/pcr-qpcr/decoded/2013/10/03/exon-numbering-not-as-easy-as-1-2-3-. [Accessed 18 Jun, 2016].

  3. Young M. (2015) Design efficient PCR and qPCR primers and probes using online tools. [Online] Coralville, Integrated DNA Technologies. Available at www.idtdna.com/pages/decoded/decoded-articles/pcr-qpcr/decoded/2015/02/27/design-efficient-pcr-and-qpcr-primers-and-probes-using-online-tools. [Accessed 18 Jun, 2016].

  4. Vandenbroucke II, Vandesompele J, et al. (2001) Quantification of splice variants using real-time PCR. Nucleic Acids Res 29(13):e68.


Product focus: Everything but your sample

All the reagents you need for successful qPCR assays are now available through IDT.

Additional resources

DECODED Online newsletter articles and technical bulletins:

  • Exon numbering—not as easy as 1, 2, 3…—Exon numbering and location data can differ across various software tools, including within NCBI's gene database. For example, exons within alternatively spliced transcripts are sometimes individually numbered, with no consistent gene-based numbering system across these transcripts for identifying exons. Here, we describe how IDT exon location information gives each exon a unique number and how that compares with the NCBI naming system.

  • Tips for using BLAST to locate PCR primers—Need a quick way to find the location of primers within a gene or the expected size of the resultant PCR product? In this tip, we show you how to get this information using BLAST.

  • Easily-designed standard curves for qPCR—Learn some of the basic concepts for designing gBlocks® Gene Fragments as standard curves or controls for qPCR.

Video tutorials

  • How to design qPCR primers spanning exon junctions using PrimerQuest—This short (~6 min) tutorial demonstrates how to use NCBI and the PrimerQuest Tool (Design across junctions) to design PCR or qPCR primers that span exon junctions for a gene. It also describes information about assay details contained in the search results.

  • Design PCR primers around a specific region with PrimerQuest—This short (~3 min) tutorial demonstrates how to use the PrimerQuest Tool (Target region list) to design PCR or qPCR primers that span a specific region of your gene.

  • Design PCR primers in a region with PrimerQuest—This short (~5 min) tutorial demonstrates how to use the PrimerQuest Tool (Excluded region list) to design PCR primers outside a specified region of a sequence, as well as re-adjusting your design parameters to find results.

  • When you need to customize PCR/qPCR primer designs—This webinar (~1 hour) provides detailed instructions for designing qPCR and PCR assays using the PrimerQuest and OligoAnalyzer tools. It also covers 3 advanced customized design scenarios: forcing a primer position (e.g., for genotyping); designing primers to span exon junctions (e.g., for gene expression); designing a probe given primer sequences (e.g., convert intercalating dye-based assay to probe-based assay).

Special guides

PrimeTime® qPCR Application Guide—Access this useful resource that provides experimental overviews, protocols, data analysis, and troubleshooting.

DECODED 4.2—Special qPCR Issue—Review an entire DECODED issue dedicated to qPCR topics, including primer and probe design, handling primer and probe oligonucleotides, qPCR using intercalating dyes, multiplex qPCR, and more.

Author: Maureen Young, PhD, is a senior scientific writer at IDT

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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