Mirneasy kit

Total RNA was isolated from harvested cells with Trizol reagent according to the manufacturer`s instructions (Life Technologies, California, USA). RNA samples were quantified using Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific) and stored at −80°C.
For miRNA analysis, total RNA including small RNA was extracted using miRNeasy Kit from Qiagen and 1μg of total RNA was used as a template to generate cDNA using miScript II RT Kit (Qiagen). To measure levels of pre-miRNAs, total RNA was extracted using miRNeasy Kit and cDNA was synthesized from total RNA (1 μg) using gene-specific primers and High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher). The gene-specific primers included a mixture of 10 μm each of the antisense primers to the selected miRNA and U6 RNA. Following an 80°C denaturation step and 60°C annealing, the cDNA was reverse transcribed for 45 min at 60°C. cDNA was diluted 1:20 before detection using Power SYBR Green PCR Kit from Applied Biosystems (ThermoFisher). Primer sequences are listed below:

Total RNA was extracted from 250 μl of ductal lavage samples using the Qiagen miRNeasy kit and the Ambion RecoverALL Total Nucleic Acid Isolation kit for FFPE, respectively, and the quantity of RNA was assessed using a Thermo Scientific NanoDrop™ Spectrophotometer.

Total RNA was extracted using either Qiagen miRNeasy Kit or Ambion mirVana miRNA Isolation kit according to the manufacturer’s instructions. Extracted RNA (10 ng per reaction) was run in duplicate in Taqman custom designed small RNA Assay and microRNA assays to determine the relative expression of sh5. cDNA was generated using a Taqman MicroRNA reverse transcription kit. Thermocycling was as follows: 16 °C 30 minutes; 42 °C 30 minutes, 85 °C 5 minutes; hold 4 °C. cDNA was diluted 1 in 5 in nuclease-free water, and 5 µl added to each custom Taqman PCR assay set up with Taqman Universal Master Mix No UNG in 20 µl reactions in 96-well plates on the Stratagene Mx3000P. Thermocycling conditions were 50 °C for 2 minutes, 95 °C for 10 minutes, 40 × (95 °C for 15 seconds; 60 °C for 1 minute).
Standard curves were generated using synthetic RNA Oligos for both sh5 and RNU38B (control gene used to normalize sh5). RNA Oligos of known copy number (10⁷–10¹) were diluted 10-fold in a background of 10 ng/μl tRNA. Data analysis was performed with Stratagene qPCR MxPro software. Expression of sh5 was determined from the sh5 standard curve and normalized to RNU38B.