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Analysis of hStaufen1-associated RNA.

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posted on 25.11.2014, 03:06 by Joan Peredo, Patricia Villacé, Juan Ortín, Susana de Lucas

Cultures of HEK293T cells were transfected with pChStaufen1-TAP (hStau) or pC-TAP (C) and soluble extracts were used for TAP purification. (A) The RNA associated was isolated from the purified complexes, 5′-end radiolabeled using γ-32P-ATP and the different RNA sizes were analysed in two denatured polyacrylamide gels, 4% (I) and 15% (II). * Indicates the size corresponding to pre-microRNAs. ** Indicates the size corresponding to mature miRNAs. The mobility of molecular weight markers is indicated to the left. (B) Small RNAs were studied using TaqMan RT-qPCR Applied multiplex to analyse 384 miRNAs with specific primers. The differences in Ct values for parallel control and hStau1 complexes are shown. Results are averages and standard deviations of 3 replicate purifications. Under the conditions used, a ΔCt of 3.3 corresponds to a ten-fold difference in RNA concentration.