# The Effect of Nerolidol Renal Dysfunction following Ischemia-Reperfusion Injury in the Rat
## 2 Materials and Methods
### Gene Expression Analysis  
A wedge from the middle part of the left kidney containing both the cortex and medulla was excised. It was then snap-frozen in liquid nitrogen and stored at \(-80\)\({}^{\circ}\)C for later measurement of gene expression of the following by reverse transcription polymerase chain reaction (RT-PCR):  
1. Acute kidney injury markers, i.e., kidney injury molecule-1 (KIM1) and neutrophil gelatinase-associated lipocalin (NGAL);
2. Cytokines involved in the inflammation and fibrosis, i.e., tumor necrosis factor-\(\alpha\) (TNF\(\alpha\)), transforming growth factor-\(\beta\) (TGF-\(\beta\)1), interleukin-6 (IL-6), interleukin-1 beta (IL-1\(\beta\)) and plasminogen activator inhibitor-1 (PAI-1);
3. The proapoptotic gene p53.
4. Procollagen type-1 (COLA-1); and
5. The antioxidant enzymes glutathione peroxidase (GPX-1) and glutathione-disulfide reductase (GSR).  
Extraction of total RNA from the frozen samples was performed using Qiazol Lysis reagent (Qiagen, Hilden, Germany) as per the manufacturer's protocol. Estimation of the quantity and quality of the extracted RNA was performed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).  
Preparation of the strand complementary DNA (cDNA) in duplicates from 1.0 ug of extracted RNA was achieved using a QuantiTect(r) reverse transcription kit (Qiagen, Hilden, Germany) as per the manufacturer's protocol. The protocol consisted of genomic DNA removal using the supplied gDNA wipeout buffer, ensuring elimination of interference by the genomic DNA. Subsequently, the prepared cDNA was used as a template for relative gene expression analysis using a TaqMan(r) hydrolysis probe chemistry kit. The reaction mixture contained 75 ng cDNA, TaqMan universal master mix (Thermo Fisher Scientific Inc., Wilmington, DE, USA), 0.5 uM of forward and reverse primers and 0.25 uM of fluorescent probe (Biosearch Technologies Inc., Petaluma, CA, USA). The probes were FAM-labeled.  
Peptidylprolyl Isomerase A (PPIA) housekeeping gene was used for normalization. Its probe was labeled with Quasar 670, enabling multiplexing with the genes of interest. All samples were run in duplicates. At least one primer of all designed PCR primer sets was spanned the exon-exon junction to further exclude any interference of the genomic DNA. Table 1 shows the sequences of primers and probes. The calculated cycle threshold (CT) values were used to estimate the changes in gene expression of target genes using a delta-delta CT formula.  
\begin{table}
\begin{tabular}{c c c} \hline \multirow{2}{*}{\
\begin{tabular}{c} KIM-1 \\ (NM\_173149.2) \\ \end{tabular} } & Forward & GCCTGAAATAATCACACTGTAAG \\ \cline{2-3}  & Reverse & GCAAACGGCACTGCCAACATAG \\ \cline{2-3}  & Probe & d FAM-TCCCTTTGAGGAAGCCGCAGA-BHQ-1 \\ \cline{2-3}  & Forward & CTGTCCCACCCGACATTCC \\ \cline{2-3}  & Reverse & CCACTGCACATCCCA \\ \cline{2-3}  & Probe & FAM-TGACAACACTGAAACAGCGTGAGCG-BHQ-1 \\ \cline{2-3}  & Forward & CTCACACTCAGATCATCTTCTC \\ \cline{2-3}  & Reverse & CCGCTTGGGTGTGTAC \\ \cline{2-3}  & Probe & FAM-CTCGAGTGAAGCCCCGTAGCC-BHQ-1 \\ \hline \multirow{2}{*}{
\begin{tabular}{c} Lippcalin 2 (LCN2) \\ (NM\_130741.1) \\ \end{tabular} } & Forward & GCCTGAAATAATCACACTGTAAG \\ \cline{2-3}  & Reverse & GCAAACGGCGCATGCAA \\ \cline{2-3}  & Reverse & GCAAACGGC  
The results are expressed as the mean fold change of gene expression compared to G-Sham. The gene expression of the G-Sham animals was measured, and the average was calculated. This average was given a value of 1. All other groups, including the G-Sham animals were compared to this value.