title: Illumina HiSeq 2500 sequencing; Comparative MicroRNA Expression Profiles of Penile Cancer Revealed by Next-Generation Small RNA Deep Sequencing
design: Comparative MicroRNA Expression Profiles of Penile Cancer Revealed by Next-Generation Small RNA Deep Sequencing
library_construction_protocol: Total RNA was extracted from ten pairs of fresh-frozen penile cancer and matched histologically normal penile tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA with good quality e.g. A260/A280>1.8 was used for further experiments. Meanwhile, the RNA integrity was evaluated by with DNA chip facilitated by Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). Two smRNA libraries were constructed using homogenized and pooled total RNAs of ten individuals for each group (normal and cancerous penile tissues). For each group, 10 microgram of total RNA was used for library construction with a Small RNA Sample Prep Kit (Illumina, USA) following the manufacturer’s instructions with minor modifications. Briefly, after 15% (w/v) denaturing PAGE (polyacrylamide gel electrophoresis), the 18- to 28-nt fraction of total RNA was excised, purified, and ligated to 3’ and 5’ RNA adaptors using T4 RNA ligase. The adaptor-ligated sRNAs were introduced to RT-PCR with 15 cycles of PCR amplification. The PCR products (approximately 90-bp, corresponding to sRNA + adaptors) were purified on 4% agarose gels to create the libraries.
attributes.tag: Experimental Factor: disease
attributes.value: penile cancer
attributes.tag: LIBRARY_STRAND
attributes.value: not applicable