Experimental Repression Targeting Long Noncoding RNAs (lncRNAs) Using an Antisense Oligonucleotide (ASO) Technology...

Area of Science:

• Dermatology and Molecular Biology
• Epigenetics and Gene Regulation
• Transcriptomics and RNA Biology

Background:

• Epidermal keratinocytes maintain skin homeostasis through proliferation and differentiation.
• Long noncoding RNAs (lncRNAs) significantly influence gene expression and cell fate but are poorly understood.
• Existing methods for studying lncRNA function have limitations in efficiency and specificity.

Purpose of the Study:

• To develop and optimize a protocol for efficient antisense oligonucleotide (ASO) delivery into primary human epidermal keratinocytes.
• To establish a reliable method for investigating the functional roles of nuclear lncRNAs in epidermal biology.
• To demonstrate the utility of ASOs for transcript-specific knockdown of lncRNAs in keratinocytes.

Main Methods:

• Optimization of antisense oligonucleotide (ASO) transfection protocols for primary human epidermal keratinocytes.
• Utilizing RNase H-mediated degradation for targeted transcript knockdown.
• Quantifying lncRNA expression levels using droplet digital PCR (ddPCR).
• Proof-of-concept knockdown of the lncRNA LINC02154 using three distinct ASOs.

Main Results:

• An optimized ASO transfection protocol was established for efficient lncRNA knockdown in epidermal keratinocytes.
• The ddPCR method provided accurate quantification of transcript expression levels post-knockdown.
• Successful repression of LINC02154 expression was achieved using ASOs, validating the approach.

Conclusions:

• ASO-mediated knockdown is a powerful and versatile tool for functional studies of nuclear lncRNAs in epidermal keratinocytes.
• This methodology facilitates a deeper understanding of lncRNA-mediated gene regulation in skin cell fate.
• The optimized protocol provides a reliable platform for advancing research in epidermal biology and skin homeostasis.