QuantiNova LNA PCR Assays for Digital PCR

Optimized digital PCR analysis with the QIAcuity EG PCR Kits and the QIAcuity instrument

QuantiNova LNA PCR Assays were developed to provide high-specificity target amplification. This can be quantified by two PCR methods: qPCR with standard real-time thermocyclers and digital PCR using the QIAcuity instrument and the QIAcuity EG PCR Kit (see figure Using QuantiNova LNA PCR Assays for digital PCR). After cDNA synthesis using the QuantiTect Reverse Transcription Kit, digital PCR provides highly precise target quantification, detecting even the smallest expression changes at the lowest concentrations. A subset of key assays have been experimentally validated on the QIAcuity instrument.

LNA enhances PCR performance

QuantiNova LNA PCR Assays have been developed using stringent design criteria and lab-validated algorithms. The rigorous assay selection process ensures that each primer set delivers the highest specificity and efficiency for the most reliable and accurate results. T_m-normalization and LNA enhancement give the primers a higher binding affinity than standard DNA primers, dramatically increasing assay sensitivity and specificity.

This increased sensitivity ensures excellent amplification efficiencies down to 1 RNA molecule, making it easier for you to detect low-abundance targets such as lncRNAs from less starting material. Increased specificity from the clever placement of LNA gives you a high signal-to-noise ratio, allowing discrimination of sequences that differ by only a single nucleotide and eliminating non-specific amplification and primer-dimer formation.

The high binding affinity of LNA bases increases the flexibility of primer placement on the transcript, so we are able to use intelligent positioning to design assays for otherwise difficult-to-analyze targets. This gives you better target discrimination and reliable quantification, even for AU-rich targets, low-abundance transcripts, targets with high secondary structure and highly complex samples.

Trust the LNA design experts

Twenty years of LNA design experience has enabled us to develop and optimize our sophisticated LNA design algorithm, which incorporates over 50 different parameters – all thoroughly lab-validated against highly stringent performance criteria – to guarantee the most optimal assay for successful target detection. We have selected up to three different assay designs for each transcript and fully wet-lab validated all popular mRNA and lncRNA target assays. QuantiNova LNA PCR Assays simply work, with no need to spend your time or money on optimization.

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 or 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.

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.

Two-step RT-dPCR

The best results are obtained when performing the reverse transcription reaction with the QuantiTect Reverse Transcription Kit (cat. nos, 205311, 205313, 205314).It is not recommended to use the QuantiNova Reverse Transcription Kit.The resulting cDNA is then quantified by dPCR using the master mixes of the QIAcuity EG PCR Kit combined with your choice of QuantiNova LNA PCR Assay.

Important note: The reaction amounts indicated in parentheses after the assay product names are relevant for qPCR use. Digital PCR only requires half the primer concentration, but reaction volumes are different. Using the QIAcuity Nanoplate 26K you can set up the same numbers of reactions as indicated; using the Nanoplate 8.5K you can set up 3.3x as many. Please refer to the handbook for QIAcuity use for dPCR instructions.

Shipping and delivery

QuantiNova LNA PCR Assays are shipped at ambient temperatures. In-stock assays are delivered within 1–5 days. During the early access period, longer delivery times should be expected.

What you need to get started for digital PCR with the QuantiNova LNA PCR system

Reverse transcription: QuantiTect Reverse Transcription Kit (cat. nos, 205311, 205313, 205314)

dPCR mastermix: QIAcuity EG PCR Kit

Assays:

• QuantiNova LNA PCR Assays
• QuantiNova LNA PCR Custom Assays

QuantiNova LNA PCR Assays are highly suited for applications including:

• mRNA and lncRNA expression analysis, profiling and quantification
• Validation of RNA-seq gene expression data
• Gene expression profiling
• Signal and pathway analysis
• Confirming gene expression knockdown by LNA GapmeRs or siRNAs
• Biomarker development, including screening, identification and validation of disease-associated biomarkers
• Monitoring phenotypic changes related to gene expression