Short oligonucleotide modalities like siRNA, miRNA, and ASO are designed to modulate gene expression by targeting mRNA, silencing or activating gene expression, or altering splicing patterns.

PCR-based technologies are the standard for bioanalytics, including biodistribution studies during cell and gene therapy development. However, short oligonucleotide modalities, including siRNA, miRNA, and ASOs, pose unique extraction, reverse transcription, and amplification challenges.

Our bioanalytical approach quantifies the short oligonucleotide, the vector (if vector-based delivery is used), the transcribed siRNA, potentially the fraction bound to the RISC complex, and the effect on the target mRNA. For biodistribution studies, this analysis is crucial in both target and non-target tissues.

PCR-based technologies, such as qPCR and dPCR, are central techniques for detecting nucleic acids due to their superior sensitivity – approximately 1000 times greater than mass spectrometry. qPCR and dPCR offer high sensitivity, specificity, and high-throughput capabilities, enabling biodistribution and transgene expression assessment in cell and gene therapies.

At TATAA Biocenter, we have developed a Two-tailed PCR technology that uses a two-tailed primer, allowing the quantification of short targets with PCR.

Assay development for PCR approaches to siRNA and ASO workflows

ASOs and siRNAs, with their various modifications, lengths, and secondary structures, perform differently during extraction and amplification. Therefore, the extraction protocol and the qPCR/dPCR assay need validation for each tissue or biofluid and test item. We develop, optimize, qualify, and validate assays tailored to each customer and context of use (COU) to accurately and reliably quantify short test items. When comparing several short test items, the extraction and PCR performance must be consistent across all constructs.

Extraction

The reliability of downstream quantification relies critically on the efficiency of the extraction process. We optimize the extraction process for each test item and biological sample, with the test item spiked in to monitor the efficiency. Modifications, construct length, chimeric oligonucleotides, and secondary structures impact extraction performance.

PCR design

Our invention, the Two-tailed PCR technology, uses a two-tailed primer that elongates the target, making it quantifiable using qPCR/dPCR. The two-tailed primer binds to the test item at two sites, offering greater specificity than the stem-loop primer, which binds at only one site. This design allows for flexibility in the binding site, the ability to discriminate between single nucleotide polymorphisms (SNPs), and optimization of PCR parameters like GC content and melting temperature (Tm), providing more opportunities for refinement.
Each custom PCR design is validated according to specified performance criteria and COU.

At TATAA Biocenter, we have developed hundreds of assays using a Two-tailed primer.