Reproducibility of miRNA Expression by Proliferating C2C12 Cells Using RTPCR Array
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abstract
RTPCR array is a new technique to study microRNA (miRNA) expression; however, minimal data regarding intra and interassay reproducibility are available. This study examined the variability of the TaqMan MicroRNA Array (Applied Biosystems) in the evaluation of RNA obtained from proliferating murine myoblast C2C12 cells. The same RNA was used for two separate reverse transcription (RT) reactions and the RT products were used in duplicate arrays. The log2 expression levels of most genes (66%) in the four replicates had a standard deviation (SD) below 0.5 with median SD=0.2. When cycle threshold (CT) exceeded 32 there was a significant SD increase from 0.23 to 1.07, thus, genes with an average CT>32 (129/381) were excluded. With CT<32, 82% of miRNA exhibited a SD<0.2. The endogenous control RNA Mammu6 correlated well in the four replicates with a coefficient of variation of 0.5% (SD<0.1) and average CT=19.6. Thus, the variability due to different RT reactions on replicate samples was low and approximately equal. We estimate that the platform can detect a fold change of 2 with 99% power and a type I error rate 0.0005, assuming 4 biological replicates under 2 conditions given the gene has an SD of 0.1. In conclusion, this realtime PCR based array method provided highly reproducibly estimates of miRNA expression by proliferating myoblasts in culture. Supported by the Veterans Administration and USPHS HL07446 and HL074236.
