7900ht sds instrument

For each sample and for each miRNA, 5 ng of total RNA was converted to cDNA by TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) with the miRNA specific primer contained in the TaqMan MicroRNA assays (Applied Biosystems). MiRNA expression was evaluated in 10 μL total volume by quantitative RT-PCR following the manufacturer protocol in the presence of 1× TaqMan Universal Master Mix (Applied Biosystems) on a 7900HT SDS instrument (Applied Biosystems). Expression differences of miRNAs among samples were determined by the comparative method according to User Bulletin #2 (Applied Biosystems) using the noncoding RNA RNU44 and RNU6B as reference gene.

For each sample and for each miRNA, 5 ng of total RNA was converted to cDNA by TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) with the miRNA specific primer contained in the TaqMan MicroRNA assays (Applied Biosystems). MiRNA expression was evaluated in 10 μL total volume in the presence of 1× TaqMan Universal Master Mix (Applied Biosystems). Taqman assays (Applied Biosystems) used for miRNA expression analysis were: 4373169 for let-7a, 4395342 for miR-9*, 4373153 for miR-10a, 4373261 for miR-20b, 4373090 for miR-21, 4395223 for miR-29a, 4373127 for miR-150, 4395459 for miR-155, 4395387 for miR-222, 4380911 for RNU44 and 4373384 for U47. QPCR analysis was performed in duplicate on 7900HT SDS instrument (Applied Biosystems) and the average cycle threshold value was used for subsequent calculations. Expression differences among samples were determined by the comparative method according to User Bulletin #2 (Applied Biosystems) using the average level of noncoding RNA RNU44 and U47 as reference.