*4.5. Reverse Transcription Quantitative Real-Time PCR*

Lymphatic vessels were homogenized in RLT and β-ME buffer using a rotating homogenizer. Cultured cells were harvested directly from culture plates with RLT-β-ME buffer and the RNA extracted using Qiagen RNeasy Mini Kit by standard protocol. Taq-Man Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) was used for reverse transcription. β-actin was used as endogenous control and fold difference in gene expression data was calculated by 2−Ct method. Human and rat NKCC1 and β-actin primers were bought from Thermo Fisher Scientific.
