Multiplexed detection of mRNA using porosity-tuned hydrogel microparticles

Area of Science:

• Biotechnology
• Molecular Biology
• Materials Science

Background:

• Transcriptional profiling is crucial for clinical prognosis and drug discovery.
• Current methods like RT-PCR indirectly quantify mRNA via cDNA amplification.
• There is a need for direct quantification of intact RNA transcripts.

Purpose of the Study:

• To develop a hydrogel microparticle-based platform for direct quantification of unmodified, full-length mRNA.
• To demonstrate the platform's sensitivity, specificity, and multiplexing capabilities.
• To show the versatility of the platform for detecting various RNA sizes.

Main Methods:

• Synthesis of poly(ethylene) glycol diacrylate (PEGDA) hydrogel microparticles using stop-flow lithography.
• Utilizing PEG600 as a porogen for efficient capture of large mRNA molecules.
• Employing a multibiotinylated universal label probe for detection.
• Demonstrating 3-plex detection using shape-encoded hydrogel microparticles.

Main Results:

• Achieved a limit of detection of approximately 6 amol for an in-vitro-transcribed model target.
• Demonstrated assay performance comparable to commercial branched DNA (bDNA) signal amplification technology.
• Successfully performed 3-plex mRNA detection without cross-reactivity.
• Showcased tunable hydrogel porosity for quantifying biomacromolecules from mRNA to miRNA.

Conclusions:

• The developed hydrogel microparticle platform enables direct, sensitive, and specific quantification of full-length mRNA.
• The platform is versatile and can be adapted for detecting a wide range of RNA molecules, including small miRNAs.
• This technology offers a promising alternative for transcriptomic analysis in various research and clinical applications.