GSM1581778: miR-seq 5; Homo sapiens; miRNA-Seq
MicroRNA samples from fresh frozen tissue were extracted using PureLink™ microRNA Isolation Kit (Life Technologies Corporation, Carlsbad, CA). Briefly, 5 mg fresh frozen tissue samples were mixed with 300 μl Binding Buffer (L3) and homogenized using a tissue homogenizer. Homogenized samples were centrifuged, and the supernatants were mixed with 300 μl 70% ethanol. Solutions containing RNA were purified by two rounds of spin column cleanup and RNA was eluted with 50 μl sterile RNase-free water. Total RNA samples were processed by the flashPAGE fractionator (Ambion) and flashPAGE Clean-Up Kit (Ambion). Enriched small RNA was then processed according to the SOLiD Small RNA Expression Kit protocol (Applied Biosystems) and purified small RNAs were ligated with 5' and 3' adapter Mix using RNA ligase. Ligated products (40–60 bases in length) were reverse transcribed and purified on Novex 10% TBE-Urea gel. Subsequently, 15–18 cycles of PCR were performed by amplifying the purified cDNA with barcoded PCR primer sets provided with the kit, which differed by a unique 6-nucleotide sequence. Amplified products were loaded on Novex 6% TBE gel (Invitrogen) and the gel bands containing 110 to 130bp fragments were excised. Amplified products were purified from the excised gel band and then loaded on the Applied Biosystems SOLiD next generation high throughput sequencing system for data acquisition. The quality of the samples and libraries were verified on the Agilent Bioanalyzer.
GEO Accession
GSM1581778