Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Low-abundance samples pose challenges for RNA analysis.
• Conventional RNA size selection methods can be inefficient and time-consuming.
• Accurate RNA size selection is crucial for downstream applications like next-generation sequencing.

Purpose of the Study:

• To develop an on-chip method for efficient RNA size selection within a specific size range (2-35 nt).
• To improve RNA extraction yield and reproducibility compared to traditional methods.
• To demonstrate the compatibility of the method with next-generation sequencing workflows.

Main Methods:

• Utilized on-chip isotachophoresis (ITP) with an ionic spacer and a sieving matrix.
• Employed ITP to separate single nucleotides and focus RNA within the 2-35 nt range.
• Compared ITP-based size selection with conventional gel electrophoresis.

Main Results:

• Achieved high yield of RNA in the target size range, excluding >90% of single nucleotides and >65% of longer RNAs.
• ITP-based selection resulted in a 2.2-fold increase in extracted target RNA compared to gel electrophoresis.
• On-chip ITP preparation led to higher reproducibility in transcript-specific measurements for sequencing.

Conclusions:

• On-chip ITP offers an efficient and reproducible method for RNA size selection from low-abundance samples.
• The method is compatible with downstream next-generation sequencing.
• ITP-based size selection is a promising alternative for analyzing small RNAs in various biological contexts.