Unique primer design for short oligonucleotides

Two-Tailed PCR technology for miRNA qPCR services

The Two-Tailed PCR is a TATAA Biocenter invention that enables qPCR and dPCR approaches to run bioanalysis on short test items such as siRNA, miRNA, and ASOs.

Advancing PCR bioanalysis

Accelerate drug development with a validated PCR assay​

PCR-based technologies are standard for bioanalytics, including biodistribution studies in cell and gene therapy development. However, short oligonucleotide modalities such as siRNA, miRNA, and ASOs present unique challenges. Our Two-Tailed PCR technology addresses these challenges by detecting the test item with high sensitivity and specificity and elongating it, thus enabling precise qPCR/dPCR analysis.The Two-Tailed PCR primer detects and extends the target in a target-specific manner during reverse transcription.

Two-tailed RT-PCR primer
TATAA Biocenter two tail

Extraction

The extraction of short oligos and chimeric oligos can be challenging. We optimize the extraction process for each test item and each tissue or biofluid because modifications, length, and secondary structure affect extraction performance.

extraction two-tail

Assay validation

The Two-Tailed primer performance is validated like any other primers in regulated bioanalysis, with the same set criteria for accuracy, precision, robustness, sensitivity and selectivity.

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How the Two-Tailed PCR Technology works

This is a highly specific, sensitive and cost-effective system is used to quantify miRNA expression based on two-step RT-qPCR, with SYBR-green detection chemistry. The results of this work can be found in the prestigious journal NUCLEIC ACIDS RESEARCH.

Two-tailed PCR
Two-Tailed PCR RT Primer

Mature microRNAs are single-stranded molecules only 22-24 nucleotides in length. They constitute a recently discovered class of non-coding RNAs that plays a key role in the regulation of gene expression. Many microRNAs are present in families that differ only in a single base position or terminal modification. Acting at the post-transcriptional level, microRNAs fine-tune the expression of many protein-encoding genes. microRNAs are present in tissues, but also in biofluids, where they are actively transported as reporter molecules. This feature makes microRNAs exceedingly attractive biomarkers and its use is currently being standardized by the European effort  SPIDIA.

There are several techniques to measure microRNA levels with quantitative PCR, but current methods use conventional pairs of primers, each 20-25 bases in length. To be able to amplify the short microRNAs they must be elongated, which often compromises sensitivity and specificity. The novel Two-tailed approach solves this problem by sensing the microRNA sequencing with two short hemiprobes. Neither hemiprobe is long enough to bind alone, but in the novel Two-tailed design the hemiprobes are connected, which leads to cooperative binding, such that either probes bind, which gives sufficient contact and stability, or neither binds. Since each hemiprobes is short, a mismatch in its binding site has a major impact, leading to the enhanced specificity of the Two-tailed method.

Two-Tailed PCR was compared to three other methods for microRNA detection is found to be more sensitive and specific, being capable to detect fewer than 10 microRNA molecules in a sample. Two-tailed PCR can be multiplexed to essentially any degree using 2-tube protocol and when using one-tube protocol multiplexing with probes is possible. Two-tailed PCR is one of the methods currently evaluated for microRNA detection in liquid biopsies collected for the management of cancer within the European effort Cancer-ID and for Sepsis within the European effort Smartdiagnos.

About us

The power of our approach

Committed to quality, innovation, and fostering strong client relationships, we serve as your trusted partner to accelerate drug development. As a contract research organization, our mission is to push the boundaries of science and technology to generate accurate and reproducible data that shortens time-to-market.

Regulated laboratory environment

We are GLP accredited for qPCR, dPCR, and molecular biology, GCLP compliant, and ISO/IEC 17025 accredited. Our facility features a strict sample management process, a fully integrated LIMS system, backup for all vital systems, temperature and humidity control, and robust IT security.

Purpose-built PCR laboratory

Our purpose-built laboratory in Gothenburg, Sweden, is specifically designed for PCR. It has controlled air pressure, temporal separation, and biosafety cabinets to minimize contamination risk. This setup enables us to achieve the highest sensitivity and robustness required for validated assays. The lab is equipped with market-leading PCR and NGS instruments and advanced liquid handling systems.

Pioneers in assay development and validation

We are a team of 45 employees, with scientists at the forefront of assay development, optimization, and validation. Our team has co-authored key publications in the field, including Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development (AAPS J. 2024) and The MIQE Guidelines for qPCR and dPCR.

Flexible, client-centered solutions

We work closely with our clients to find tailored solutions, offering flexibility in sample types, test items, sample volume, and scalability. We prioritize transparency and proactive communication throughout each project, ensuring our clients are continuously updated on the progress.

Quantitative PCR (qPCR), digital PCR (dPCR), and Next-Generation Sequencing (NGS) are essential technologies in siRNA bioanalysis. They provide the precise quantification, sensitivity, and specificity needed to track siRNA’s therapeutic efficacy, stability, and biodistribution. Application note: siRNA assays in bioanalysis (PDF)

Small RNAs, such as siRNA, miRNA, snoRNA, and others ranging from approximately 18 to 26 nucleotides, cannot be amplified using conventional PCR primers, as a single primer matches their entire length. Various techniques are used to elongate short RNA molecules

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