SP fraction analysis and miRNA profiling of clinical prostate tissues.

Abstract

<p>(A) Human prostate cancer tissue was converted to single cell suspensions as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040021#s2" target="_blank">Methods</a>’. Cells were stained with Hoechst dye and analyzed as described previously. Nearly 1% of total viable cell population was gated as the SP fraction. (B) Total RNA was isolated from another set of human prostate tissues (cancer and benign) using miRNEasy RNA isolation kit. SYBR Green based pathway-focused miScript miRNA PCR Array (Qiagen, MD) was used for miRNA profiling studies. The plates were run on a Roche Light Cycler 480® instrument and the expression of individual miRNAs was analyzed using the obtained C_t values and the ΔΔCt method. The fold changes in the tumor tissues were normalized with respect to the benign prostate tissue. (C) Table in the insert confirms validation of miRNA profiling data by miScript primer assay. Validation of miRNA profiling data was done by RT-PCR estimation of selected miRNAs200c, 34a and 29b. SNORD6 was used as a housekeeping miRNA for data normalization.</p