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Comparative analysis of AMOUR and other strategies for profiling surface RNAs

ABSTRACT: Conventionally limited to proteins, lipids, and polysaccharides, cell membranes have recently revealed the presence of protein-coding RNAs and glycan small RNAs on the surface of mouse and human cells. Despite glycan-RNA's prevalent presence on the cell exterior, a comprehensive method for accurately mapping all surface RNAs, particularly for rare cell populations has been lacking, leaving their physiological roles largely unexplored. We developed AMOUR, an RNA capture technique coupled with T7-based linear amplification, enabling precise profiling of surface RNAs in mammalian cells while preserving plasma membrane integrity.To ensure both sensitivity and specificity, we developed two supplementary strategies: WGA-Pd and RT-Amp, for surface RNA profiling alongside AMOUR.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252682 | GEO | 2025/12/02

REPOSITORIES: GEO

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Project description:Background: Insufficient quantities of human genomic DNA are a limiting factor for many clinical applications. Whole genome amplification (WGA) is an approach designed to overcome small amount of DNA for genome-wide genetic tests as it allows amplification of the entire genome from picogram or nanogram quantities of DNA. Various strategies of WGA have been developed; however, none of them can guarantee the absence of amplification bias. High-quality genome-representative amplified DNA is crucial for WGA use in basic research and clinical genetics. Thus, systematic evaluation of WGA effect on downstream methods is necessary. Results: In this paper, 4 multiple displacement amplification (MDA) -based and 2 PCR-based WGA kits were compared in their effect on segmental copy-number changes as well as copy-number neutral loss of heterozygosity detection by high-density oligonucleotide DNA arrays. We described outcomes and limits for each individual WGA; however, the main goal of this study was chiefly to show a general compatibility and features specific for particular WGA strategy. The main outcomes are as follows: 1) MDA-based WGAs showed higher tendency to generate false positive imbalances in contrast to PCR-based WGAs with higher risk of false negativity; 2) the specific risk of false positivity and/or negativity increased with decreasing copy-number segments size; 3) single-cell WGAs showed significantly worse effect on results in comparison to WGAs with nanogram level of DNA as input; 4) PCR-based WGAs were not compatible with copy-number neutral loss of heterozygosity analysis based on single nucleotide polymorphisms in restriction digestion sites and also showed higher risk of copy-number neutral loss of heterozygosity false negativity if combined with analysis based on simple hybridization. Conclusions: This study gives a comprehensive insight into the WGA effect on DNA array analysis. The results of this study help to choose WGA according to individual user requirements and options. Moreover, we show a strategy to verify and validate segmental copy-number changes detection by DNA array protocol including any WGA for any purpose to attain the highest efficiency without an unnecessary WGA bias.

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Comparative analysis of AMOUR and other strategies for profiling surface RNAs

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