Quantitative PCR (qPCR) is the method of choice for precise quantification of gene expression. qPCR can utilize a variety of probe-based methods such as 5′ nuclease dual-labeled probes, molecular beacons, FRET probes, and Scorpions™ Probes, or use intercalating fluorescent dyes such as SYBR. 5′ nuclease assays have the advantage of the specificity that comes with using a sequence-specific, dual-labeled probe, and is the preferred technique for gene expression analysis. This article will focus on 5′ nuclease assay design and experimental setup considerations that will assist in obtaining accurate and consistent results.
A well-designed assay begins with an understanding of the gene of interest, including knowledge of the transcript variants and their exon organization. Following are recommendations for obtaining the optimum assay designs for qPCR gene expression analysis.
Know your gene
Use NCBI databases such as GenBank, Gene, and dbSNP to identify exon junctions, splice variants, and SNP locations.
- For genes with multiple transcript variants, align related transcripts to understand exon overlap using a program such as ClustalW, or any of the NCBI online tools.
- For splice-specific designs, target primers and probe(s) within exons unique to the transcript(s) of interest, and BLAST primer and probe sequences to ensure they do not occur in other related transcripts (see the article, Tips for using BLAST to locate PCR primers).
- For splice common designs, target primers and probe within exons found across all transcript variants.
- Ensure lack of cross reactivity with other genes within the species.
Primer and probe design criteria
Once the target exons have been identified for design, use the following criteria to select primers and probe.
- Tm: Primer Tm values should be similar (+/-2°C). For 5′ nuclease qPCR assays, Tm values are normally approximately 60−62°C.
- Length: Aim for 18−30 bases in length. This length typically yields a Tm of ~60−62°C.
- GC content: Avoid runs of >4 Gs to prevent formation of G quadruplexes. GC content should range from 35−65% (ideally, ~50%).
- Sequence: Avoid sequences that may create secondary structures, self-dimers, and heterodimers. Use the free IDT online OligoAnalyzer 3.1 Tool to identify any such structures.
- Location—minimize genomic DNA amplification: If measuring gene expression, prevent amplification of genomic DNA by designing primers to span exon-exon junctions. Alternatively, design primers within 2 adjacent exons spanning a large intron (Figure 1). Under normal qPCR cycling conditions, amplification of this large PCR product would not be favored. A DNase treatment step can also be incorporated to eliminate gDNA amplification.
- Location—avoid repeated sequences: BLAST potential primer sequences and redesign them when they cross react in multiple places in the genome. Also consider primer location relative to SNPs. SNPs underlying the 3' primer end will have an impact on amplification.
Figure 1. Primer design to limit genomic DNA amplification.
- Tm: For optimal detection of amplification, the probe Tm value should be 4−10°C higher than that of the primers. A probe with a higher Tm ;than the primers should be annealed to the template as amplification begins.
- Length: Limit probe length to 30 bases when using dual-labeled probes designed with most common quenchers, as beyond this length quenching ability is decreased. However, you can design longer probes if you include an internal quencher, such as the IDT ZEN™ quencher (For more information, see the article, Decrease qPCR Background, Improve qPCR Signal).
- GC content: To prevent G quadruplex formation, minimize the number of consecutive Gs. GC content should range from 30−80%.
- Sequence: Avoid a G base at the 5′ end as it has been observed that G bases can have a quenching effect on dyes such as FAM™ and other fluorescein derivatives.
- Location: Probes can be designed to bind to either the sense or antisense strand.
Length: For typical cycling conditions, ideal amplicon size is between 70 and 200 bp. Longer amplicons can be designed, but cycling conditions should be adjusted to include longer extension times. Generally, slightly longer amplicons are used for SYBR-based assays than for probe-based assays to enable differentiation from primer dimers on a melt curve.
Experimental setup and controls
For accurate analysis of qPCR results, each experiment needs to be set up with multiple replicates and controls (Figure 2).
Replicates: For each experimental and control sample to be compared, at least three technical replicates are necessary to minimize errors in measured gene expression due to pipetting.
Figure 2. qPCR assay setup. Outline of the test and controls needed for an experiment with two different samples examined at several time points post treatment. Include 3–5 biological replicates for each time point studied. For each biological replicate studied, perform 2 reverse transcription reactions (+RT) and 1 with no RT (–RT control). For each cDNA sample generated, set up 3 technical replicates for qPCR analysis. Include a No Template Control for each gene analyzed to identify any signal due to contamination.
- No RT control: For every reverse transcription reaction, it is important to incorporate a “no RT control” to identify erroneous signal due to genomic DNA contamination.
- No template control: During qPCR setup, incorporate a “no template control” for every different gene analyzed to identify possible cross-contamination generated during preparation of samples.
- Reference genes: Multiple reference genes should be analyzed for stable gene expression. Select a reference gene with an expression pattern that is unaltered by treatment or the different time points to be tested.
- Cycling conditions: Always follow the cycling conditions according to the master mix used. For example, do not use a standard master mix with fast cycling conditions.