Droplet digital PCR (ddPCR), an emerging analytical technique in recent years, overcomes the lack of sensitivity and accuracy of traditional PCR, which is suitable for the detection of trace nucleic acids in exosomes and the analysis of copy number variant status. Creative Biolabs is committed to developing an advanced platform for exosomal components analysis and provides Exosomal RNA Digital PCR Analysis services.

Overview of ddPCR

ddPCR, also known as "water-in-oil PCR", involves the micro-dropping of the sample prior to conventional PCR amplification, whereby the reaction system containing nucleic acid molecules is divided into thousands of nano-sized droplets, each of which contains one to several nucleic acid target molecules to be detected. After PCR amplification, each microdroplet is detected one by one, the microdroplet with fluorescence signals is interpreted as 1 while that without fluorescence signals is interpreted as 0. The starting copy number or concentration of the target molecule can be obtained according to the Poisson distribution principle and the number and proportion of positive microdroplets. ddPCR technology not only effectively improves the amplification efficiency of nucleic acid molecules, but also fundamentally improves the quantification of nucleic acid molecules by using microdroplet counting. The technique breaks through the technical limitation of quantification based on fluorescence intensity and allows absolute quantification of nucleic acid molecules in the measured samples. The detection sensitivity of digital PCR has been improved by 10-100 times compared to ordinary RT-PCR, with a more than 10 times reduction in sample usage.

Exosomal RNA Digital PCR Analysis Service at Creative Biolabs

Based on the absolute advantage of digital PCR in terms of sensitivity, it is especially suitable for the detection and validation of ng-level micro samples like exosomal RNA. When the amount of exosomal RNA is small, digital PCR can perform stable detection at 0.1 ng, while the detection starting amount of common RT-PCR is usually around 1 ng, which makes it difficult to complete the detection of multiple genes. Creative Biolabs has incorporated digital PCR into the sequencing validation and independent detection experiments of exosomal RNAs and established a rigorous and reliable technical route, which supports the acquisition of reliable digital PCR data.

Fig.1 Schematic diagram of exosome RNA digital PCR analysis procedure at Creative Biolabs. (Creative Biolabs)

Application of Exosomal RNA Digital PCR Analysis

Due to the low levels of exosomes in the peripheral circulation, accurate and reliable analysis of the nucleic acids contained therein relies on highly sensitive assays. ddPCR allows for rapid, accurate, and absolute quantification of gene expression levels in exosomes. For example, a Taqman probe-based ddPCR technique has been developed and applied to detect the expression of PML-RARA fusion genes in exosomes derived from NB4 cell culture supernatants and in exosomes derived from the serum of APL patients. This provides a non-invasive, rapid and absolutely quantifiable method for dynamic monitoring of small residual lesions of the disease. Furthermore, the combination of a target-specific identification system and ddPCR for quantification of PD-L1 in exosomes enables differentiation between exosomes of tumor and healthy donor origin. This approach combines significant selectivity and accuracy, facilitating the conversion of exosomes into more reliable diagnostic indicators of tumors.

Fig.2 Working principle of the highly sensitive quantification of tumor-derived Exo-PD-L1 using aptamer-based PLA. (Lin, 2021)

ddPCR is an advanced precision nucleic acid quantification technique for the determination of gene transcript expression in exosomes, giving the possibility of non-invasive, real-time, and more reliable monitoring of diseases. Creative Biolabs offers exosomal RNA digital PCR analysis service, which is highly sensitive and has excellent reproducibility. Please contact us to discuss your project.

Reference

1. Lin, B.; et al. Tracing tumor-derived exosomal PD-L1 by dual-aptamer activated proximity-induced droplet digital PCR. Angew Chem Int Ed Engl. 2021, 60(14): 7582-7586.