Quantitative Analysis of Oligonucleotides: The Strength of RT-qPCR

Reverse tranion-quantitative polymerase chain reaction (RT-qPCR) exhibits unique advantages in the quantitative analysis of oligonucleotides due to its ultra-high sensitivity, wide linear range, high throughput, and low sample consumption. This blog introduces principles, advantages, challenges, and solutions of relevant RT-qPCR techniques and shares some insights based on the experience of WuXi AppTec DMPK.

What is RT-qPCR?

qPCR is recognized as one of the standard methods for quantifying nucleic acids harboring known sequences. RT-qPCR is usually used for quantitative analysis of RNA. RNA is first transcribed into complementary DNA (cDNA) and qPCR is then carried out to amplify cDNA and quantitation.

Oligonucleotides typically consist of 19 to 25 bases, which are too short for designing primers and probes using conventional methods to amplify nucleotide sequences. To address this challenge, several methods for lengthening the test nucleic acid sequence have been developed, including the stem-loop RT-qPCR, Poly(A) polymerase tailing-based RT-qPCR, ligation-based qPCR, and primer-extension qPCR. Among these, the stem-loop RT-qPCR and Poly(A) polymerase tailing-based RT-qPCR are more commonly used.

What are the advantages of RT-qPCR for oligonucleotide quantitative analysis？

• Ultra-high detection sensitivity and wide dynamic linear range：For the detection of plasma samples in low-dose groups or long experimental periods, a highly sensitive detection method is necessary. RT-qPCR offers a wide dynamic linear range for quantification, typically over 7–8 orders of magnitude, facilitating the bioanalysis of high- and low-abundance samples. The ultra-high sensitivity of RT-qPCR makes it possible to detect the RISC-loaded siRNA, providing information for intracellular delivery of siRNA and the mechanism of RNA silencing.

  
  

• Enabling high throughput sample analysis and reducing the use of biological samples：During preparing RT-qPCR samples, applying high-throughput and high-precision automated liquid handling systems to construct the experimental systems and reaction system allows for avoiding inhomogeneity, reducing experimental errors, and improving sample processing efficiency. Additionally, RT-PCR requires only approximately 10 µL of biological samples, thereby reducing the use of biological samples.

What are the challenges and solutions in using RT-qPCR for oligonucleotide quantification?

• Primer and probe design: It is necessary to prevent the formation of primer-primer and primer-probe dimers, which may lead to non-specific amplification or abnormal results. In addition, it is important to ensure that the Tm values of primer pairs are as close as possible, and the Tm value of the probe is 5-10℃ higher than that of the primer.

• Harsh operating environment and consumable requirements: It is necessary to strictly avoid aerosol contamination and interference from nucleases during the experimental process.

• Metabolites may affect method accuracy: qPCR lacks perfect specificity for oligonucleotide analytes in biological samples, which may affect the accuracy.

• Chemical modifications or carriers may affect sensitivity and accuracy: Unique optimization steps are necessary for some oligonucleotides that may reduce the detection sensitivity of RT-qPCR, such as changing the ion concentration in the reaction system.

• Lack of guidance from regulatory agencies: Validation of qPCR/RT-qPCR assays is not currently required by the FDA or NMPA, and there are no uniform standards for experimental design, evaluation processes, and acceptance criteria for sample analysis.

WuXi AppTec DMPK Non-GLP Bioanalysis Team has established an assay system for qPCR/RT-qPCR method development and validation. In addition, we have developed an RT-qPCR bioanalytical method with high sensitivity and excellent accuracy and precision for quantitative analysis of oligonucleotides in biological samples during screening, preclinical PK analysis, and the establishment of PD/PK modeling.

If you want to learn more details about the application of RT-qPCR in DMPK quantitative analysis of oligonucleotides, please read the article now.

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