Authors
Yessenia Magaña, Shanshan Wang, Albertina Torreblanca-Zanca, Lai Wei, Michael J Castle
Published in
eNeuro. Jul 24, 2026. Epub Jul 24, 2026.
Abstract
Adeno-associated virus (AAV) vectors are widely used for neuroscience research. Labeling AAV mRNA by in situ hybridization can track AAV transduction with high sensitivity and without the need for reporter genes. However, it is challenging to detect similar mRNA transcripts when multiple AAVs are injected into the same animal, or when a single AAV produces multiple distinct transcripts. To address these challenges, we developed methods for sensitive labeling of small barcodes on AAV mRNA at single-transcript resolution. These small RNA barcodes (ranging from 40 to 44 bases) can be easily added to any AAV vector genome. We provide detailed step-by-step protocols for sensitive and flexible labeling of barcoded AAV mRNA by BaseScope in situ hybridization. We validated these labeling methods in male and female mice, and in a female rhesus macaque. RNA barcoding can improve the reliability of multiplexed in vivo screening: by adding one barcode to half of the AAVs in a pooled library and a second barcode to the other half, barcode labeling can identify cells that receive multiple AAVs without the need for reporter proteins. This approach is also useful for intersectional experiments that combine multiple AAV vectors: multiple unique barcodes can track the spatial distribution of each AAV and can separately label transcripts from different AAV genome conformations, such as before or after Cre-Lox recombination. These new methods for sensitive multiplexed labeling of AAV mRNA will support greater rigor and reliability in neuroscience research.Significance Statement AAV vectors are commonly used in neuroscience research. Modern experimental designs are increasingly complex, often injecting two or more AAVs into different regions, or screening a library of many unique AAVs injected together into a single animal. In such complex experiments, it is challenging to track spatial distribution and rates of co-infection among multiple AAV vectors, especially if the AAVs do not carry reporter genes. To address these challenges, we developed small RNA barcodes and methods for highly sensitive labeling of barcoded AAV mRNA. These small barcodes can be easily added to any AAV genome without altering the experimental design, supporting the assessment of injection targeting and co-infection rates among multiple AAVs without the need for reporter genes.
PMID:
42498665
Bibliographic data and abstract were imported from PubMed on 25 Jul 2026.
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