Ulf Landegren

Enhanced detection and distinction of RNA by enzymatic probe ligation

It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe–target mismatch by a factor of between 20- and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.

Additional file 9: Figure S8. of Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction

PCA separation of the various time points (0 hr = purple, 24 hr = green, 48 hr = blue). a A PCA over all quantitative protein targets and the corresponding ROC curves (b) for all three time points generated from random forest decision categorization with AUC of 0.98, 0.94, and 0.86 for 0 hr, 24 hr, and 48 hr, respectively. c A PCA over all quantitative RNA targets and the corresponding ROC curves (d) for all three time points generated from random forest decision categorization with AUC of 0.81, 0.80, and 0.57 for 0 hr, 24 hr, and 48 hr, respectively. e A PCA over all quantitative protein and RNA targets and the corresponding ROC curves (f) for all three time points generated from random forest decision categorization with AUC of 0.99, 0.94, and 0.84 for 0 hr, 24 hr, and 48 hr, respectively. For a, c, e, axis labels indicate which PC was used and what percent variance it explains. (PDF 269 kb)