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. 2015 May 4;43(11):5601–5616. doi: 10.1093/nar/gkv432

Figure 6.

Figure 6.

Validation of the sequencing results by SL-RT-ddPCR and SL-RT-qPCR, and purification of ribonucleoprotein complexes containing 5′ tRNA halves. (A) 2D-plots showing the relative abundance (Log2 copies per nanograms of input RNA) of selected miRNAs and tRNA-derived fragments between extracellular and intracellular fractions, measured by droplet digital PCR (ddPCR). Light gray, dark gray and white dots correspond to p16, p100 and S100, respectively. (B and C) Increased relative abundance (measured by ddPCR (B); qPCR (C)) of 5′ tRNA halves derived from tRNAGlyGCC (left; 1–30 nt) and tRNAGluCUC (right; 1–31 nt) in S100 (gray bars) with respect to the intracellular compartment (diagonal pattern). Abundances of tRNA halves were normalized to miR-21–5p and miR-16–5p. Significantly higher normalized abundances of tRNA halves in S100 versus INTRA are shown (Student t test; one-tailed). (D) Protease and RNase protection assays of the p100 and S100 fractions. ΔCq values were calculated between samples not treated and treated with RNaseA. The other experimental variable was treatment with Proteinase K (NT: not treated; +PRK: treated). Three biological replicates of each condition were analyzed, and each replicate was assayed for miR-21–5p and 5′ halves of tRNAGlyGCC and tRNAGluCUC (thus, each bar corresponds to nine different ΔCq values). (E) Top: The S100 fraction from MCF-7 cells was injected in a Superdex 75 10/300 column (size-exclusion chromatography) and fractions (200 μl) were eluted in 1× PBS using an ÄKTA purifier FPLC system (GE Healthcare, Amersham). The chromatograms correspond to the absorbance at 280 nm (gray area with dashed contours) and 260 nm (black line). V′R: adjusted retention volume (VR - Vm). Bottom: Eluted fractions were assayed for the presence of 5′ halves from tRNAGluCUC (gray; left Y axis) and tRNAGlyGCC (black; right Y axis) by SL-RT-qPCR. The horizontal gray dashed line corresponds to the input (before injection) 2−Cq value for tRNAGluCUC. The input 2−Cq value for tRNAGlyGCC was 7.5×10−8.