Disruption and homogenization of the tissue sample is performed in binding buffer, Buffer TM1. After centrifugation to remove residual cell debris, isopropanol is added to the lysate to provide appropriate binding conditions for all RNA molecules 18 nucleotides and longer. The sample is then applied to a PAXgene RNA MinElute spin column, where total RNA binds to the membrane and contaminants are efficiently washed away. Between the first and the second wash step, the membrane is treated with DNase I to remove trace amounts of bound DNA. After the wash steps, RNA, including miRNA, is eluted in a low-salt elution buffer and denatured by heating.
Tissues from different organs were fixed for 3 hours in PAXgene Tissue Fix, transferred to PAXgene Tissue Stabilizer, and stored for 24 hours at room temperature. After processing, total RNA was purified from sections of paraffin-embedded tissue using the PAXgene Tissue RNA Kit (RNA/miRNA). miRNA-enriched fractions (200 nt) were also isolated from sections of the same tissue blocks using the PAXgene Tissue miRNA Kit (Enriched miRNA). Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-10a using the miScript Reverse Transcription Kit and the miScript SYBR® Green PCR Kit.