Technologies → Short RNA PCR

Highly sensitive PCR assays for short RNA and DNA

TATAA Biocenter’s unique Two-Tailed PCR technology enables qPCR/dPCR method development, validation, and high-throughput analysis of siRNA, miRNA, ASOs, and gRNA.

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Tailored, regulatory-compliant

qPCR and dPCR assays for siRNA, miRNA, ASOs, and gRNA.

A more flexible and efficient solution for

siRNA, miRNA, gRNA and ASO PCR than stem-loop primers

Short nucleic acids such as siRNA, miRNA, gRNA, and ASOs are too small to be directly amplified by conventional PCR, which requires two primers. The short target must be elongated during the cDNA synthesis step to enable PCR-based bioanalysis, allowing for validated method development.

Two-tailed PCR primers offer greater design flexibility, making them more adaptable for optimization. This flexibility increases the likelihood of meeting assay criteria for a validated method.

References
Androvic, P. et.al. Two-tailed RT- qPCR panel for quality control of circulating microRNA studies. Scientific Reports – Nature 2019, 9:4255.

Androvic, P. et. al. Two-tailed RT-qPCR: A novel method for highly accurate miRNA quantification. Nucleic Acids Res. 2017, 45, e144.

How the Two-Tailed PCR technology works

Small RNA analysis with qPCR and dPCR

Primer design

A hairpin primer with two target-specific hemiprobes enables precise detection.

Specificity

Cooperative binding enhances specificity.

Target elongation

Reverse transcriptase elongates the target, making it compatible with standard PCR amplification.

PCR bioanalysis

Quantification with standard qPCR or dPCR.

qPCR and dPCR assay characterization

Category	Feasibility	Method Development	Method Qualification	Method Validation
Validation or sample analysis study plan		Optional	Required	Required
Assay Design	3 Designs per target	3 Designs per target	3 Designs per target	3 Designs per target
Assay Efficiency and Amplicon Confirmation	√	√	√	√
Establish calibration curve parameters	√	√	√	√
Linear Range of Assay		√	√	√
Accuracy and Precision in surrogate matrix (buffer)		2 runs, 5 levels, 3 sets
Extraction Efficiency		3 kits, 1 level of spike, QC of nucleic acids Quality and Integrity, 1 repeat	1 kit, 3 levels of spike, QC of nucleic acids Quality and Integrity, 2 repeats	1 kit, 3 levels of spike, QC of nucleic acids Quality and Integrity, 2 repeats
Minimum Required Dilution (MRD)		√	√	√
Accuracy and Precision in authentic matrix		2 runs, 5 levels, 3 sets	3 runs, 5 levels, 3 repeats	6 runs, 5 levels, 3 repeats
Selectivity and Specificity in authentic matrix		Optional	√	√
Dilution linearity		Optional	√	√
Stability		Optional	Optional	√
Data QC		20%	50% or 100%	100%
QA involvement			Optional	√
Report		√	√	√
Acceptance criteria		Depending on Context of Use	Depending on Context of Use	Depending on Context of Use
QC levels		LLOQ, LQC, MQC, HQC, ULOQ	LLOQ, LQC, MQC, HQC, ULOQ	LLOQ, LQC, MQC, HQC, ULOQ

GLP for qPCR and dPCR

We are accredited for Good Laboratory Practice (GLP) by the Swedish Board for Accreditation and Conformity Assessment (SWEDAC) for qPCR, dPCR, and molecular biology.

Short RNA/DNA PCR services at TATAA

Samples

Tissues
Blood
Liquid biopsies
Fresh frozen
FFPE
EDTA
PAXgene tubes

Analytes

miRNA
ASO
siRNA
gRNA
Short viral RNA

How stem-loop primers work

A stem-loop primer is used in RT-PCR for short RNA molecules such as siRNA, miRNA, gRNA, and ASOs. It elongates the target RNA during cDNA synthesis, enabling quantification via qPCR or dPCR similar to the Two-tailed PCR.

The difference lies in primer design flexibility and the ability to optimize the primer to meet regulatory assay criteria. A stem-loop primer is simple to design since it has only one configuration: a segment hybridizes to the last six to eight nucleotides at the 3′ end of the target RNA. However, this provides limited optimization options for primer specificity and performance. If a stem-loop primer delivers the required specificity, sensitivity, and robustness for a validated assay, it can be suitable for siRNA, ASO, gRNA, and miRNA bioanalysis.