An integrated microfluidic system for automated extraction of in vitro transcribed mRNAs via probe-coated magnetic...

Extraction and purification of mRNA are critical steps for diverse molecular biological applications, including gene expression analyses, vaccine development, and genomic assays. To enhance mRNA extraction efficiency, we designed an integrated microfluidic chip capable of isolating in vitro transcribed (IVT) mRNAs (up to 93%) via probe-coated magnetic beads without the need for high-speed centrifugation, organic solvents, or cooling conditions. mRNA-specific probe-coated magnetic beads were employed, thereby automating the entire workflow, including IVT, DNase treatment, mRNA extraction, and reverse transcription (RT) could all be performed on-chip. This advancement is particularly significant for on-chip transcription-translation coupled with puromycin linker association (TRAP) display screening applications. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed the device's ability to selectively capture mRNA with a high capture efficiency of 93% within 15 minutes and 48% of thermal release efficiency after 10 minutes at 95 °C. By incorporating this optimized approach into a fully automated lab-on-chip system, puromycin-linked mRNAs could be directly used for peptide screening of kinesin family member 2C (KIF2C), a protein overexpressed in aggressive cancers.