RNA extraction was performed from 200 μl of plasma samples. miRNAs were isolated using Total RNA Purification Plus Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer's protocol, as previously described [39 (link)]. As an internal control, 10 ftmoles of cel-miR-39a was spiked into each plasma sample after lysis.
RNA from skin biopsies was extracted with an RNeasy tissue lipid kit (Qiagen, Valencia, CA, USA), as previously described [40 (link)]. miRNA expression levels in skin samples were normalized to housekeeping RNA Z30 expression.
miRNA levels were analysed using the TaqMan quantitative real-time PCR (qRT-PCR) and quantified with the QuantStudio5 real-time PCR (Thermo Fisher Scientific, Massachusetts, United States). Primers for miR-200c, miR-200a, miR-200b, miR-429, miR-141, Z30, and cel-miR-39a and the reagents for reverse transcriptase and qPCR reactions were all obtained from Applied Biosystems. miRNA expression levels in each sample were normalized to cel-miR-39a. Relative expression in fold was calculated using the comparative Ct method (2–ΔΔCt) [41 (link)].
Given the logarithmic nature of the q-PCR CT, a decrease in ΔCT corresponds to an increase in miRNA levels in the analysed samples. Therefore, data were expressed as −ΔCT, in order to obtain a positive correlation between miRNA levels and clinical parameter values.
RNA from skin biopsies was extracted with an RNeasy tissue lipid kit (Qiagen, Valencia, CA, USA), as previously described [40 (link)]. miRNA expression levels in skin samples were normalized to housekeeping RNA Z30 expression.
miRNA levels were analysed using the TaqMan quantitative real-time PCR (qRT-PCR) and quantified with the QuantStudio5 real-time PCR (Thermo Fisher Scientific, Massachusetts, United States). Primers for miR-200c, miR-200a, miR-200b, miR-429, miR-141, Z30, and cel-miR-39a and the reagents for reverse transcriptase and qPCR reactions were all obtained from Applied Biosystems. miRNA expression levels in each sample were normalized to cel-miR-39a. Relative expression in fold was calculated using the comparative Ct method (2–ΔΔCt) [41 (link)].
Given the logarithmic nature of the q-PCR CT, a decrease in ΔCT corresponds to an increase in miRNA levels in the analysed samples. Therefore, data were expressed as −ΔCT, in order to obtain a positive correlation between miRNA levels and clinical parameter values.
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