Analysis artefacts of the INS-IGF2 fusion transcript

Area of Science:

• Molecular Biology
• Genomics
• Immunology

Background:

• Gene expression analysis is susceptible to artefacts from overlapping genes, splice variants, and fusion transcripts.
• The INS-IGF2 fusion transcript was previously reported as highly expressed in human beta cells and implicated in Type 1 Diabetes.

Purpose of the Study:

• To investigate the true abundance of the INS-IGF2 fusion transcript in human beta cells.
• To identify the source of artefacts leading to overestimation of INS-IGF2 expression.
• To validate the presence of INS-IGF2 protein using available antibodies and proteomics.

Main Methods:

• RNA sequencing
• Variant-specific quantitative PCR (qPCR)
• Western blotting
• Immunohistochemistry
• Proteomics analysis

Main Results:

• The true abundance of INS-IGF2 is over 20,000-fold lower than INS in human beta cells.
• A commercial antibody (BO1P) against INS-IGF2 showed significant cross-reactivity with native proinsulin.
• INS-IGF2 protein was not detected in human beta cell lines or islets via Western blotting, immunohistochemistry, or proteomics.

Conclusions:

• Fusion transcripts like INS-IGF2 can cause significant artefacts in gene expression analysis.
• The abundance of INS-IGF2 transcript and protein is exceedingly lower than previously reported.
• Current immuno-reagents for INS-IGF2 detection exhibit cross-reactivity with proinsulin, and INS-IGF2 protein was not detected by sensitive proteomics.