Copy number determination of sperm-borne small RNAs implied in the intergenerational inheritance of metabolic syndromes

Copy number determination of specific 5′ tDRs in murine spermatocytes and oocytes. (A) Representative northern blot of a mass titration series (250-5 pg) of unmodified 5′ tDR-Gly^GCC and 5′ tDR-Glu^CUC along with total RNA (50 ng) extracted from spermatocytes of mice (n = 3) using a probe against the 5′ moiety of tRNA-Gly^GCC and 5′ tRNA-Glu^CUC, respectively. (B) Representative northern blot of a mass titration series of unmodified 5′ tDR-Ala^AGC, -His^GUG, and -Lys^CUU (500-5 pg) alongside total RNA (50 ng) extracted from spermatocytes of mice (n = 3) using a probe against the 5′ moieties of the parental tRNAs. (C) Boxplots summarizing the calculated copy number of specific 5′ tDRs from different tRNA isoacceptors and one rsRNA (rsRNA-28S-1) per spermatocyte in different individual mice (N above individual boxplots). The median (black horizontal line) and the mean (red diamond) are marked. The individual blots and their analysis, resulting in the displayed data points, are described in the Supplemental Tables S2–S4. (D) Representative northern blotting of a mass titration series of unmodified rsRNA-28S-1 (500-5 pg) alongside total RNA (50 ng) extracted from spermatocytes of mice (n = 5) using a probe against the 28S rRNA. (E) Northern blotting of total RNA (50 ng) extracted from spermatocytes of mice (Sp, n = 2) and total RNA extracted from two pools of germinal vesicle oocytes (Oo, n = 100, Roman numerals) alongside a mass titration series of unmodified 5′ tDR-Gly^GCC and 5′ tDR-Glu^CUC (500-5 pg) using a probe against the 5′ moieties of tRNA-Gly^GCC and 5′ tRNAs-Glu^CUC, respectively. For this blot, two tDR mass titration series were loaded subsequently into the same lanes (unmodified 5′ tDR-Gly^GCC followed by unmodified 5′ tDR-Glu^CUC) along with RNA from biological samples, which was loaded at the time of loading unmodified 5′ tDR-Gly^GCC. The blot was first probed for tRNA-Gly^GCC followed by stripping and re-probing for tRNA-Glu^CUC. Black arrowheads: mature tRNAs; red arrowheads: 5′ tDRs; empty arrowheads: tDRs or rsRNA-28S-1 mass titration series, respectively; blue arrowheads: rsRNAs-28S-1.