Quantitative PCR Data Analysis

Raw PCR Ct values were generated using QuantStudio™ Real-Time PCR Software with automatic baseline and threshold (Thermo Fisher). The data set was uploaded to Thermo Fisher Data Cloud and further analyzed using Thermo Fisher Cloud Software-Relative Quantification. PCR Ct values ≥ 35 were considered as undetectable and excluded from further analysis. An appropriate internal normalizer could not be identified due the composition of the different subcellular sources of RNA. Therefore, the Global Mean Normalization method [54 (link)] was used to normalize the TLDA or single-tube data. The relative miRNA levels or Gapdh mRNA quantity (RQ) in each fraction were expressed as 2^−∆Ct value (2 to the power of negative deltaCt).