(A) piRNA piR-1 (5′-TGAAGACGGACAGAAGATGGGTTAATTATTT-3′) expression was validated using Northern blotting of early stage oocytes (stages I and II). One hundred and fifty oocyte equivalents were used to analyze small RNA expression. A miR-101 probe was used as a control. β-Elimination was performed to assay for 2′O-methyl-modified 3′ nucleotides. EDC was used to crosslink small RNAs to the membrane prior to hybridization. (B) Experiments were performed as in A, but with probes for piR-2 (5′-TGAATTGTAGAACAATGTACAGGTACACCAT-3′) and miR-202-5p. (C) Expression of piR-1 and miR-148a was assessed in a number of adult somatic tissues and early stage oocytes (stages I and II) using Northern blotting. Ten micrograms of total RNA were used for each lane. 5S rRNA is shown as a loading control. (D) Northern blot analysis of immunoprecipitated piRNAs using piwil1.1/piwil1.2 specific antibody (PI: pre-immune).
