Mir 223

cDNA was generated from 1 μg of isolated mRNA from ectocervical cells using the miScript II Reverse Transcription kit (miRNA) (Qiagen) for SYBR Green. qPCR was performed on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Life Technologies) using the miScript SYBR Green PCR kit (Qiagen) according to the manufacturers’ protocol. The ΔΔCT method was used for relative expression quantification using the QuantStudio Real-time PCR software v1.1 (Applied Biosystems, Life Technologies). The endogenous reference gene RNU6B was used for miRNA quantification. All miRNA primer sets were purchased from Qiagen: miR-143 (MS00003514), miR-145 (MS00003528), miR-193b (MS00031549), miR-146a (MS00003535), miR-223 (MS00003871), miR-148b (MS00031458), miR-15a (MS00003178), miR-21(MS00009079), miR-142 (MS00031451), miR-494 (MS00033754), miR-30e (MS00009401), and RNU6B (MS0001400).

RNA from the cells was isolated with the RNAeasy mini kit (Qiagen, Hilden, Germany), and cDNA synthesis was performed. PCR was performed using qPCR master mix containing SYBR green with primers of GAPDH and pro-inflammatory molecules (IL-1β, IL-18, NLRP3) (Applied Biosystems 7500 Fast, Foster City, CA, USA) (Table 2). For the detection of miRNAs, miR-223 and endogenous control U6 primer assays were utilized (Qiagen, Hilden, Germany). The expression difference was analyzed by the ΔΔCt method with endogenous normalization to GAPDH or U6 [23 (link)].