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.
Most comprehensive and specific coverage
Our proprietary algorithm has been used to design over 1.3 million QuantiNova LNA PCR Assays to provide the most sensitive, accurate and effective mRNA and lncRNA analysis. The predesigned assays cover most transcripts in the Ensembl database for human, mouse and rat genes, enabling PCR-based gene expression studies in the greatest depth possible. Most of the assays are intron-spanning when possible and detect only RNA. Assays that do not span an intron are designated as such, and if there is one exon in the target, unwanted signals can be easily eliminated using the QuantiTect Reverse Transcription Kit with the integrated gDNA removal step.
Choosing the right assay for your target
Predesigned QuantiNova LNA PCR Assays let you accurately and sensitively detect any human, mouse or rat mRNA or lncRNA, no matter what level of analysis you need: general transcript detection, detection of a specific transcript or differentiation of transcript isoforms.
Most human, mouse and rat genes have only one transcript, but for those with multiple transcripts, we selected up to three assays per transcript using the same algorithm. The design and primer positioning of these assays differ to match various usage requirements. Our assay selection guide helps you quickly identify the best assay from each category. After searching for assays for your target, simply review the results and look for the recommended assay that matches your intended use:
- Best coverage: for general transcript detection
- Best transcript assay: for detection of a specific transcript
- Best transcript-specific assay: for differentiation between specific transcript isoforms
Refer to the table below for more details.
|QIAGEN’s recommended assay
||Marked as “Best coverage”
||Marked as “Best transcript assay”
||Marked as “Best transcript-specific assay”
|Description of assay design and coverage
||Covers most of the biologically relevant transcripts of the given gene
||Highly optimized to specifically detect the transcript of interest, targeting more isoforms of the transcript
||Splice variant transcript-specific
|When to choose
||For general transcript screening and determining whether the gene of interest is expressed in a sample; for detection of as many transcripts and isoforms of a gene as possible
||For detection of a specific transcript
||For differentiation between specific isoforms of a transcript
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.
Reference Gene Assays for any study
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 from real-time PCR experiments. 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.