Unique primer design for short oligonucleotides
Two-Tailed PCR Technology
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.
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.
Assay validation
The performance of the RT step, primers, and probes for qPCR/dPCR is validated according to set performance criteria and COU. If multiple test items are to be compared, the assay is designed so that all constructs are amplified equally effectively.
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.
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.
Services
Solutions for complex biological challenges
We specialize in nucleic acid therapy drug development and bioanalysis, extracting and detecting your test item in biological samples with the necessary performance requirements, whether regulated or non-regulated. Additionally, we perform biomarker profiling on small non-coding RNAs, messenger RNA, genome, and protein.
technologies
We operate with the latest technologies
TATAA Biocenter comprises highly skilled scientists with extensive expertise in method development, data analysis, and optimization across diverse technologies. We leverage top-notch instrumentation and automated platforms, offering expert guidance to choose the most suitable technology on the market for your program.
PCR
We use highly sensitive and highly precise quantitative PCR (qPCR) and digital PCR (dPCR) technologies to detect one or multiple DNA/RNA targets in complex samples.
NGS
We use automated, customized workflows tailored to the sample type to simultaneously analyze thousands of genes, whole genomes, and either targeted RNA or full transcriptomes for both small and large-scale studies.
Quantitative proteomics
Olink’s proximity extension assays (PEA) allow multiplexed analysis of hundreds to thousands of proteins using just microliters of blood or plasma, ideal for immune studies and biomarker discovery or validation.
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
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
contact
Let's explore how we can support your drug development program.
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