RT Journal Article SR Electronic T1 Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs JF bioRxiv FD Cold Spring Harbor Laboratory SP 068031 DO 10.1101/068031 A1 Yun S. Choi A1 Lanelle O. Edwards A1 Aubrey DiBello A1 Antony M. Jose YR 2016 UL http://biorxiv.org/content/early/2016/08/05/068031.abstract AB 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 C. 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 a ~360-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 equivalent detection of 24 nt to 14 nt RNAs. Our protocol can better discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans. This improved northern blotting is particularly useful to obtain a measure of small RNA integrity, analyze products of RNA processing or turnover, and analyze functional RNAs that are shorter than typical miRNAs.