*4.4. Analyses*

#### 4.4.1. Gene Expression Analysis of IVD Tissue

RNA extraction from IVD tissue and the following steps were performed according to [19]. For cDNA synthesis, two micrograms of RNA were used in a total volume of 60 μL, while using the reverse transcription kit (4374966, Applied Biosystems, Foster City, CA, USA). For samples with lower yields, the reverse transcription was conducted at reduced concentrations. cDNA (10 ng/well) was mixed with TaqMan Fast Universal PCR Master Mix and TaqMan primers (Table 6) to quantify gene expression. The obtained *C*<sup>t</sup> values were analyzed by a comparative method and displayed as 2−d*C*<sup>t</sup> values, with GAPDH as housekeeping gene.
