Quantification of AQP5 mRNA in Septic Patients

The RNA samples collected from whole blood were considered eligible for analysis when their concentration was greater than 50 ng/µl, according to nanodrop measurement (ND-1000, PEQLAB Biotechnologie GmbH, Erlangen, Germany). The RNA samples from 98 septic patients were available in acceptable quality. First-strand cDNA was synthesized from 0.5 µg of total RNA using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The qPCR reaction was performed using GoTaq® qPCR Master Mix (Promega, Mannheim, Germany), as described previously^{5 (link)}. A cDNA dilution series for AQP5 confirmed a PCR efficiency >95%, which was comparable to the efficiency of ß-actin (data not shown). Relative AQP5 mRNA expression was measured by two-step qPCR and expressed as 2^-∆CT.