Brilliant 2 sybr master mixes

Total RNA from glomeruli, LMD samples, cultured cells, and exosome fractions was isolated using an miRNeasy Mini Kit (Qiagen; Venlo, Netherlands). Culture medium was collected and centrifuged at 21,000×g for 10 min, and total supernatant RNA was isolated using a 3-fold volume of Trizol LS (Life Technologies). To determine mRNA levels in glomeruli and cultured cells, total RNA was used as template to synthesize cDNA using reverse transcriptase (RT). Quantitative PCR analysis was performed using Brilliant II SYBR Master Mixes (Agilent) and specific primers (Table S4) with an MX3000P system (Agilent). The specificity of each PCR reaction was confirmed using melting curve analysis. The expression data were normalized to the expression levels of Actb (tissues) or Gapdh (cells). miRNA levels were determined using a TaqMan MicroRNA RT Kit (Applied Biosystems; Foster City, CA, USA). Quantitative PCR analysis was performed using each miRNA-specific TaqMan primer and TaqMan Universal PCR Master Mix (Applied Biosystems) with an MX3000P system (Agilent). We determined the levels of U6 snRNA for data normalization.

2μg of total RNA was retro transcribed with AffinityScript cDNA Synthesis Kit (Agilent Technologies, Milano, Italy) following the manufacturer's instructions. Specific primer sets (Supplementary Table S1A) were designed with Primer3 (http://www.broad.mit.edu/cgi-bin/primer/primer3) to amplify 100–200bp products. cDNAs were diluted to a final concentration of 20 ng per reaction. Real-time quantitative rtPCR was performed in triplicate using Brilliant II SYBR® Master Mixes (Agilent Technologies) on Mx3005P™ Real-Time PCR System (Agilent Technologies) and expression values were normalized against α-tubulin mRNA.

To generate cDNA with the AffinityScript cDNA Synthesis Kit (Agilent Technologies, Rome, Italy), 1 μg of total RNA was used. cDNAs were diluted to a final concentration of 20 ng per reaction. Real-time qRT-PCR was performed in triplicate using Brilliant II SYBR Master Mixes (Agilent Technologies) on an Mx3005P Instrument (Agilent Technologies); the expression level of PIWIL genes was normalized against β-actin mRNA. Specific primer sets are reported in Table 1.