EvaGreen-based digital PCR
For absolute quantification by dPCR, use the QuantiNova LNA PCR Assays together with the QIAcuity EG PCR Kit, which uses EvaGreen-based fluorescence detection. EvaGreen is an intercalating dye that binds to double-stranded DNA (similarly to SYBR® Green) and fluoresces upon DNA binding. Using EvaGreen-based dPCR provides convenience and savings, because you only need the primer set to amplify and detect the product. It also provides higher resolution in the dPCR reaction, which is divided into thousands of partitions of the dPCR nanoplate, so the primer concentration needed for the dPCR reaction is only half that required for qPCR.
The custom design process
The QuantiNova LNA PCR Custom Assay design tool lets you easily design highly sensitive and specific, LNA-enhanced PCR assays for any mRNA or lncRNA not available as a predesigned assay. Using the advanced QuantiNova PCR Assay design algorithm, numerous assay combinations are evaluated based on more than 50 different criteria to find the optimal assay for your target within a few minutes. The tool has been designed for human, mouse and rat mRNA and lncRNA targets of at least 55 nucleotides and blasts against the species-relevant databases, including Ensembl and RefSeq. For known gene targets, assays are designed as intron-spanning by default, similar to the predesigned assays, to prevent the risk of gDNA amplification.
Uniquely advanced assay design options
The custom assay design tool includes options to create transcript-specific designs for discriminating splice variants, SNPs or isoforms. Alternatively, designs for assays common to all transcript variants can also be created. You can submit up to 10 different target sequences at once and design either ten different assays, or for related transcripts, you can design a common assay for all.
Reference Gene Assays can be easily combined with the custom assays
A wide selection of functionally validated human, mouse and rat Reference Gene Assays are available to enable high-quality data normalization and ensure reliable results. These assays target endogenous coding RNAs, long non-coding RNA and small nucleolar RNA molecules that are typically constitutively expressed in a wide variety of tissues.
Normalization of mRNA/lncRNA qPCR results
Normalization removes technical and biological inter-sample variation unrelated to the biological changes under investigation. Proper normalization is critical for correct analysis and interpretation of results. Most commonly, stably expressed reference genes are used for normalization.
It is generally recommended to test several endogenous control reference gene candidates before setting up your actual mRNA/lncRNA expression analysis. These candidates should be chosen from genes expected to be stably expressed over the whole range of samples under investigation. They could be stably expressed mRNAs or lncRNAs selected based on literature or preexisting data (e.g., NGS or qPCR panel screening). The QuantiNova LNA PCR system offers validated reference gene assays for RNAs that tend to be stably expressed and are therefore good candidates as reference genes.
All reference gene candidates should be empirically validated for each study. One option for normalizing PCR panel when profiling a large number of mRNAs/lncRNAs is to normalize against the global mean – the average of all expressed mRNAs/lncRNAs. This can be a good option in samples with a high call rate (expressed genes) but should be used with caution in samples with low call rates. It is also not a good option in samples for which the general gene expression level is changed. Further guidance on normalization can also be found in the GeneGlobe Data Analysis Center.
Digital PCR data analysis
The QIAcuity Software Suite is used for analyzing the dPCR data and includes a gene expression test, which provides your results as fold change and fold regulation with publication-ready figures.