Quantification of piR-24000 Expression

Expression of piR-24000 within the tissue specimens was analyzed by reverse transcription followed by SYBR-green-chemistry-based quantitative PCR. Universal oligo dT primer (for reverse transcription) and amplification primers (for quantitative PCR) were designed as per Balcells et al. [44 (link)]. For the reverse transcription reaction, 100 ng of total RNA was polyadenylated using E. coli Poly(A) Polymerase (New England Biolabs, Massachusetts, United States), and was subsequently reverse transcribed using the PrimeScript RT Reagent Kit (Takara, Kyoto, Japan) as per the manufacturer’s guidelines. Quantitative estimation of piR-24000 was subsequently carried out by the ABI VIIa PCR system (Applied Biosystems, California, United States) using the TB Green Premix Ex Taq II (Takara, Kyoto, Japan) kit. All experiments were performed in duplicate and results were normalized to the expression of RNU6B and expressed as −∆Ct (negative delta Ct). Primer sequences used are listed in Supplementary Table S2.