Circulating MicroRNAs in Patients with Vulvar Squamous Cell Carcinoma and its Precursors

Data table contain the values of the relative miRNAs expression. 

Total RNA was isolated from 200 µL of plasma using the miRNeasy Serum/Plasma Kit (Qiagen).  100 ng of total RNA were used for miRNA reverse transcription using Pool A of Megaplex™ RT Human Primers (Applied Biosystems) in the Veriti™ 96 Well Thermal Cycler (Applied Biosystems). The miRNA cDNA was then preamplified using Megaplex™ PreAmp Primers, Pool A, in the Gene Amp® PCR System 9700 (Applied Biosystems). Following dilution, preamplified microRNA cDNA was loaded onto TaqMan® MicroRNA Arrays A v2.0 (Applied Biosystems) containing 384 TaqMan® MicroRNA Assays per card. The qRT-PCR amplification and data acquisition were performed on the ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific|Applied Biosystems). These microfluidic cards allow the quantification of 377 human microRNAs. The reverse transcription, pre-amplification, dilution, and qPCR steps were performed according to the manufacturer’s protocols (Applied Biosystems). 

The collected data were analyzed using threshold-cycle (Ct) values for the miRNAs with the Relative Quantification (RQ) Application Module on the Thermo Fisher Cloud using automated baseline and manually set threshold values. The results were normalized with endogenous control included in the TaqMan MicroRNA Arrays. Ct values were exported and the relative amounts of each miRNA in the plasma of patients were calculated using the 2−ΔCtT method. U6 snRNA was selected for qPCR data normalization