QuantiNova LNA Probe PCR Assays provide highly robust gene expression analysis using hydrolysis probe-based detection. Choose from the broadest selection of predesigned assays for accurate and sensitive detection of any human, mouse or rat mRNA or lncRNA – from general transcript detection to differentiation of specific transcript isoforms. LNA enhancement allows us to design shorter primers and probes, which are more flexible to position on the target. As a result, we can optimize our designs for specific transcript detection or target differentiation. Plus, thorough design validation ensures optimal performance and robust detection, and assays for all popular mRNAs and lncRNAs undergo wet-lab validation. Optimized using QuantiNova chemisty, the simple QuantiNova LNA Probe PCR workflow can be completed in under 2 hours and is robust enough to support room-temperature setup, automation and delayed run of assays.
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QuantiNova LNA Probe PCR Assays have been developed using stringent design criteria and lab-validated algorithms. The rigorous assay selection process ensures that each primer–probe set delivers the highest specificity and efficiency for the most reliable and accurate results. Tm-normalization and LNA enhancement give the primers and probe a higher binding affinity, dramatically increasing assay sensitivity and specificity.
This increased sensitivity of QuantiNova LNA Probe PCR Assays 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 (see figure QuantiNova LNA Probe PCR Assays provide accurate, sensitive and linear quantification of targets over a wide dynamic range and figure QuantiNova LNA Probe PCR Assays enable both high-expression and low-expression detection of mRNA). 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 and probe 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 robust and reliable quantification, even for AU-rich targets, low-abundance transcripts, targets with high secondary structure and highly complex samples.
QuantiNova LNA Probe PCR Assays were developed to give optimal results and the highest reproducibility when used with our trusted QuantiNova PCR chemistry. For 1-step qRT-PCR, this includes the QuantiNova Probe RT-PCR Kit. For 2-step qRT-PCR, this includes the QuantiNova Reverse Transcription Kit and the QuantiNova Probe PCR Kit (see figure Demonstrated lot-to-lot reproducibility).
The QuantiNova hot-start mechanism enables successful qPCR, even for difficult templates, such as those with high GC-content or complex secondary structure – ideal for lncRNA analysis (see figure Consistently high efficiencies throughout the expected GC content range of the human transcriptome). The hot start also allows reaction setup at room temperature, and is robust enough to support automated setup and delayed run of assay plates. Plus, the built-in visual pipetting color indicator helps prevent human pipetting errors.
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 Probe PCR Assays simply work, with no need to spend your time or money on optimization.
The fast and easy workflow can be completed in under 2 hours with minimal hands-on time and can be automated to save even more time and labor. The simple protocol has been tested for compatibility on all major brand qPCR cyclers (see figure QuantiNova LNA Probe PCR Assays deliver consistent performance across all thermal cycler instruments). Furthermore, the cDNA generated in the RT step can be used across the entire QuantiNova LNA Probe PCR System, allowing seamless transition from assays to panels depending on your research needs, saving you time and sample.
QuantiNova LNA Probe PCR Assays use FAM-labeled hydrolysis probe-based detection, which is highly sensitive and robust, because only the desired PCR product is detected. Probe-based detection still requires high PCR specificity to prevent amplification artifacts such as non-specific PCR products and primer–dimers that can compete for reaction components and compromise performance. However, we’ve already optimized the QuantiNova LNA Probe PCR system for you to eliminate non-specific amplification and give you accurate and sensitive detection every time.
Our proprietary algorithm has been used to design over 1.3 million QuantiNova LNA Probe 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 QuantiNova Reverse Transcription Kit with the integrated gDNA removal step.
Predesigned QuantiNova LNA Probe 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:
Refer to the table below for more details.
|Gene-covering assays||Transcript-specific assays|
|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|
The complimentary, web-based data analysis tool in the GeneGlobe Data Analysis Center includes a user-friendly wizard to guide you step-by-step through the normalization and analysis of your data and generates publication-ready results figures.
A wide selection of 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. The Reference Gene Assays are FAM labeled and have been functionally validated as reference genes for the QuantiNova LNA PCR system and work optimally with the QuantiNova RT and PCR reagents.
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. Further guidance on normalization can also be found in the GeneGlobe Data Analysis Center.
QuantiNova LNA Probe PCR Assays have been optimized using the time-tested and high-performance QuantiNova chemistry. Best results are obtained when reverse transcription is performed with the QuantiNova Reverse Transcription Kit. The resulting cDNA is then quantified by qPCR using the master mixes of the QuantiNova Probe PCR Kit combined with your choice of QuantiNova LNA Probe PCR Assay.
For 1-step qRT-PCR, we recommend using the QuantiNova Probe RT-PCR Kit, which offers quick reaction setup without need for optimization of reaction conditions. Simply add primers, probe and RNA template to the ready-to-use RT-PCR master mix and start the reaction.
QuantiNova LNA Probe PCR Assays are shipped on dry ice. In-stock assays are delivered within 1–5 days. During the early access period, longer delivery times should be expected.
Reverse transcription: QuantiNova Reverse Transcription Kit
qPCR mastermix: QuantiNova Probe PCR Kit
One-Step qRT-PCR (optional): QuantiNova Probe RT-PCR Kit
Assays or panels:
QuantiNova LNA Probe PCR Assays are highly suited for applications including: