Materials and Methods
Cells and Stimulation
The immortalised BEC line H69 was cultured to confluence. Resting cells were compared against stressed cultures that had 200 μM H2O2 added to the medium for 2 h; the cells were subsequently washed and returned to normal medium for 4, 8, 12, 24 and 36 h. RNA was isolated from resting and stressed cells using TRI reagent (Sigma).
Cell Fixation and Paraffin Embedding
Three cell pellets were created: resting cells, 12-hours post-H2O2 injury and 24 hours post-H2O2 injury. Cells were suspended in 10 mL of 10% neutral buffered formalin overnight at 4°C. The cells were then centrifuged and the pellets embedded in paraffin.
Tissue Samples
Six times zero (post-reperfusion) FFPE liver transplant biopsy specimens were used in this study. These tissue samples were from separate patients transplanted before 2006 in accordance with Research Ethics Committee approval (06/Q0905/150).
FFPE mRNA Isolation and Validation
RNA was isolated from FFPE liver tissue using an RNeasy FFPE kit (Qiagen). For each reaction, 4×10 μm sections were freshly cut from the FFPE blocks; the outer 40 μm was discarded to minimise the effects of oxidation. The purity and quantity of each RNA sample was determined by Nanodrop spectrophotometry; RNA integrity was validated by agarose electrophoresis (50 mL TAE buffer, 0.5 g agarose and 3 μL Ethidium Bromide) and an AlphaImager (Alpha Innotech) to identify the 18 S and 28 S subunits of ribosomal RNA.
cDNA Synthesis and TaqMan Real-time PCR
The RNA was reverse transcribed to cDNA using an AffinityScript Multi-Temperature cDNA synthesis kit (Agilent Technologies). TaqMan chemistry was used with exon-spanning primers (Applied Biosystems) and a StepOne Plus real-time PCR system (Applied Biosystems) to quantify gene expression. The cycle threshold (Ct) for genes of interest (p21/WAF1, transforming growth factor (TGF)-β1 and TGF-β2) was normalised using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase to allow calculation of fold changes in gene expression.