Removing bias against short sequences enables northern blotting to better complement RNA-seq

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, University of Maryland rsearchers 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. They 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, they 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, the researchers greatly reduced this bias to enable equivalent detection of 24 nt to 14 nt RNAs. This 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.

A subset of miRNAs is detected predominantly with missing nucleotides
at the 5’ end in some small RNA-seq datasets

(A) Many miRNAs show altered average lengths in two different RNAseq datasets. The average length of miRNAs detected in RNA-seq datasets both from wild-type animals (black) and from mut-16(-) (magenta), mut-2(-) (blue), or wild-type animals undergoing pos-1 RNAi (green) were determined. The average length of each miRNA was normalized to that detected in the dataset from wild-type animals and plotted in rank order starting with the miRNAs that show the most proportional shortening. Total numbers of miRNAs present in both datasets (shared miRNAs) for each pair of datasets compared are indicated. (B) A common set of miRNAs, including the entire miR- 51 to 56 family, show the most shortening in two different RNA-seq datasets. The average lengths of the most shortened miRNAs detected (A) were plotted for all datasets. Dataset labels as in (A) and the miR-51 to 56 family (brown) is highlighted. (C) The shorter length of miRNAs in both datasets is due to a shorter 5’ end and is specific to the more abundantly sequenced arm of the miRNA. 1000 RNA reads that map to a miRNA (miR-52 shown as an example) were sampled from wild-type (black), mut-2(-) (blue), or wild-type animals undergoing pos-1 RNAi (green) and the resultant coverage (reads per base) of both arms of the miRNA gene (-5p and -3p, which are also in numbers per 1000 5p reads in insets) were plotted.