ArticleNucleic acids research2017
Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs.
Article in Nucleic acids research, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 49 citations in OpenAlex.
- Detection of tRNA-Derived Small RNAs in Arabidopsis thaliana via Northern Blotting: Exploring Potential Biomarkers for Phytohormone Analysis.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Isothermal and protein-free cascade catalytic hairpin assembly induced-DNAzyme sensing strategy for sensitive miRNA analysis.Scientific reports · 2025Article
- Article
- Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.Biosensors & bioelectronics · 2021Article
- Endogenous microRNA triggered enzyme-free DNA logic self-assembly for amplified bioimaging and enhanced gene therapy via in situ generation of siRNAs.Journal of nanobiotechnology · 2021Article
- Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods.BMC genomics · 2019Article
- Photoelectrochemical biosensor for microRNA detection based on multiple amplification strategies.Mikrochimica acta · 2018Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm Caenorhabditis elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using northern blotting during these studies, we discovered that miRNA-length probes can have ∼1000-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable nearly equivalent detection of 24 to 14 nt RNAs. Our protocol can discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans and pRNAs in total RNA from bacteria. This improved northern blotting is particularly useful to analyze products of RNA processing or turnover, and functional RNAs that are shorter than typical miRNAs.
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