Why should I use dPCR to analyze RNA targets?

Digital PCR delivers true molecular counts of RNA or lncRNA targets. Unlike qPCR, dPCR is independent of reference curves and requires no normalization using a housekeeping gene, which increases the reliability of cross-sample comparisons. Because of its partitioning strategy and end-point readout dPCR detects very low numbers of RNA or lncRNA molecules in a sample, outperforming qPCR for rare or low-expression transcripts, such as many lncRNAs.

What are the benefits of using locked nucleic acids (LNA)-modified primers for detecting mRNA and lncRNA with dPCR?

Primers with higher binding affinity and increased melting temperature, enabling very short or specific primers for the detection of low-abundance RNA targets even in complex or degraded samples Enhanced ability to distinguish transcript isoforms or single nucleotide polymorphisms, enabling differentiation among highly similar RNA species or mutant vs wild-type transcripts Successful amplification from challenging biological samples, like serum, plasma, biofluids, or FFPE tissues, where RNA is often low-quality or present in trace amounts

Which species are supported with the mRNA/lncRNA digital PCR assays?

The pre-designed assays cover human, mouse, and rat mRNA/lncRNA targets.

What’s the difference between mRNA and lncRNA digital PCR assays?

mRNA dPCR assays focus on well-characterized transcripts. The primary goal of these dPCR tests is to accurately capture expression with a clean, short amplicon. lncRNA dPCR assays are more complex because the typical targets are of low abundance and overlap with other genes. Often, you need more stringent, LNA-enhanced designs to ensure specific and sensitive detection.

What are predesigned mRNA digital PCR assays?

Predesigned mRNA digital PCR assays on GeneGlobe are ready-to-use QuantiNova LNA PCR Assays that have been optimized for sensitive and accurate detection of human, mouse and rat mRNA targets. Each assay is bioinformatically validated for specificity, short amplicon length and LNA-enhanced performance, allowing reliable quantification of gene expression without the need for custom design. Popular targets are also wet-lab verified. This means that for commonly studied genes and transcripts, the assays have been experimentally tested in QIAGEN labs to confirm their performance (specificity, sensitivity and reproducibility).

How can I design a custom digital PCR assay for mRNA targets?

Depending on your application, if no predesigned assay is available, you can create custom dPCR assays for gene expression analysis using our custom assay design tools or contact our Genomic Services team for additional support.

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Digital PCR assay design tips	RNA targets	lncRNA targets
Amplicon length	Keep amplicons short (<150 bp) to minimize secondary structure	Short amplicons (<150 bp) are preferred, especially due to fragmentation in complex matrices or low-abundance targets
Primer design	Use primers against spliced regions for mRNA Design primers with balanced GC content and minimal secondary structure Consider specificity if targeting isoforms	LNA-modified primers/probes can improve binding specificity, especially for challenging lncRNA targets where sequence similarity and secondary structure can cause false amplification Custom primer/probe design is often required for unique or overlapping transcripts
Probe design	Hydrolysis probes (TaqMan-type) are preferred for multiplexing and specificity Probes should avoid G at the 5′-end to minimize quenching Design junction-specific probes for editing or splicing events	LNA enhancement increases mismatch discrimination and signal-to-noise for lncRNA detection Consider a design to distinguish sense/antisense or overlapping lncRNAs from mRNA targets
Specificity and cross-reactivity	Use BLAST or similar to avoid non-specific binding Design junction probes for spliced RNA Consider the need to distinguish similar isoforms	Stringent cross-reactivity control is needed due to frequent genome overlap and similarity to protein-coding genes and pseudogenes LNA designs and multiple probe sets are beneficial for lncRNA specificity
Assay optimization	Optimize annealing temperature, Mg2+, and primer/probe concentration, especially for detection sensitivity.	Same steps as for RNA, but lncRNA is often low abundance – may require enhanced detection chemistry, increased input and more stringent RT controls.
Controls	Use RT controls (minus RT to confirm RNA specificity), positive controls and endogenous normalizers (e.g., RPP30, GAPDH) for normalization.	Use synthetic spike-ins, matched normalizers (from stable lncRNA or external standard) and sample-to-sample variability controls.

Digital PCR assay design tips

Amplicon length

Keep amplicons short (<150 bp) to minimize secondary structure

Short amplicons (<150 bp) are preferred, especially due to fragmentation in complex matrices or low-abundance targets

Primer design

Use primers against spliced regions for mRNA
Design primers with balanced GC content and minimal secondary structure
Consider specificity if targeting isoforms

LNA-modified primers/probes can improve binding specificity, especially for challenging lncRNA targets where sequence similarity and secondary structure can cause false amplification
Custom primer/probe design is often required for unique or overlapping transcripts

Probe design

Hydrolysis probes (TaqMan-type) are preferred for multiplexing and specificity
Probes should avoid G at the 5′-end to minimize quenching
Design junction-specific probes for editing or splicing events

LNA enhancement increases mismatch discrimination and signal-to-noise for lncRNA detection
Consider a design to distinguish sense/antisense or overlapping lncRNAs from mRNA targets

Specificity and cross-reactivity

Use BLAST or similar to avoid non-specific binding
Design junction probes for spliced RNA
Consider the need to distinguish similar isoforms

Stringent cross-reactivity control is needed due to frequent genome overlap and similarity to protein-coding genes and pseudogenes
LNA designs and multiple probe sets are beneficial for lncRNA specificity

Assay optimization

Optimize annealing temperature, Mg2+, and primer/probe concentration, especially for detection sensitivity.

Same steps as for RNA, but lncRNA is often low abundance – may require enhanced detection chemistry, increased input and more stringent RT controls.

Controls

Use RT controls (minus RT to confirm RNA specificity), positive controls and endogenous normalizers (e.g., RPP30, GAPDH) for normalization.

Use synthetic spike-ins, matched normalizers (from stable lncRNA or external standard) and sample-to-sample variability controls.

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Digital PCR assays for RNA targets

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dPCR Microbial DNA Detection Assays

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How to use dPCR to detect RNA targets and lncRNA targets

Compare dPCR assay design considerations for RNA vs lncRNA and get practical tips for each in the table below.

Digital PCR assay design tips

RNA targets

lncRNA targets

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FAQ – Digital PCR assays for RNA analysis