Gene Expression Analysis Using Liver Transplant Biopsy
Gene Expression Analysis Using Liver Transplant Biopsy
Experiments in vitro showed that oxidative stress induced a significant increase in the expression of p21/WAF1, TGF-β1 and TGF-β2 by BEC (figure 1; all p<0.001), with maximal upregulation observed after 12, 12 and 4 h, respectively. The RNA extracted from these cells before and after oxidative stress for 4 and 12 h (figure 2A) showed intact 18S and 28S ribosomal RNA subunits with no evidence of degradation. However, although the RNA-extracted FFPE cell pellets at these time points had a reasonable purity (typical A260/280=2.03 indicating little protein contamination, and A260/230=2.02 indicating little solvent contamination), none of these samples showed discrete 18S or 28S bands (figure 2B). Gene expression analysis of these samples showed a significant increase in both p21/WAF1 and TGF-β1 12 h after oxidative stress (figure 3; both p<0.001). However, no expected increase was observed in expression of the TGF-β2 transcript.
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Figure 1.
Measurement of gene expression by cultured human biliary epithelial cells at various times after 2 h of oxidative stress (***p<0.001). Summary data of three separate experiments are shown.
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Figure 2.
Representative gel showing RNA extracted from non-fixed (A) and formalin-fixed, paraffin-embedded processing (B) biliary epithelial cells; non-degraded 18S and 28S ribosomal RNA bands can be seen in panel (A).
(Enlarge Image)
Figure 3.
Measurement of gene expression by cultured human biliary epithelial cells at various times after a brief period of oxidative stress and following formalin-fixed, paraffin-embedded processing (***p<0.001). Summary data of three separate experiments are shown.
A quantity (2.95±0.42 μg; mean±SEM) of pure RNA (260/280 and 260/230 ratios were all above 2) sufficient for cDNA production was isolated from sections of each of the six FFPE liver biopsy samples. After reverse transcription, each sample showed exponential amplification during real-time PCR generating measurable C t values. One of the six cases was arbitrarily chosen to normalise the summary data presented in figure 4.
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Figure 4.
Gene expression analysis using formalin-fixed, paraffin-embedded biopsy-derived RNA from six liver transplant cases; all biopsy samples were taken at time zero immediately after reperfusion. All results were normalised to one arbitrarily chosen sample.
Results
Experiments in vitro showed that oxidative stress induced a significant increase in the expression of p21/WAF1, TGF-β1 and TGF-β2 by BEC (figure 1; all p<0.001), with maximal upregulation observed after 12, 12 and 4 h, respectively. The RNA extracted from these cells before and after oxidative stress for 4 and 12 h (figure 2A) showed intact 18S and 28S ribosomal RNA subunits with no evidence of degradation. However, although the RNA-extracted FFPE cell pellets at these time points had a reasonable purity (typical A260/280=2.03 indicating little protein contamination, and A260/230=2.02 indicating little solvent contamination), none of these samples showed discrete 18S or 28S bands (figure 2B). Gene expression analysis of these samples showed a significant increase in both p21/WAF1 and TGF-β1 12 h after oxidative stress (figure 3; both p<0.001). However, no expected increase was observed in expression of the TGF-β2 transcript.
(Enlarge Image)
Figure 1.
Measurement of gene expression by cultured human biliary epithelial cells at various times after 2 h of oxidative stress (***p<0.001). Summary data of three separate experiments are shown.
(Enlarge Image)
Figure 2.
Representative gel showing RNA extracted from non-fixed (A) and formalin-fixed, paraffin-embedded processing (B) biliary epithelial cells; non-degraded 18S and 28S ribosomal RNA bands can be seen in panel (A).
(Enlarge Image)
Figure 3.
Measurement of gene expression by cultured human biliary epithelial cells at various times after a brief period of oxidative stress and following formalin-fixed, paraffin-embedded processing (***p<0.001). Summary data of three separate experiments are shown.
A quantity (2.95±0.42 μg; mean±SEM) of pure RNA (260/280 and 260/230 ratios were all above 2) sufficient for cDNA production was isolated from sections of each of the six FFPE liver biopsy samples. After reverse transcription, each sample showed exponential amplification during real-time PCR generating measurable C t values. One of the six cases was arbitrarily chosen to normalise the summary data presented in figure 4.
(Enlarge Image)
Figure 4.
Gene expression analysis using formalin-fixed, paraffin-embedded biopsy-derived RNA from six liver transplant cases; all biopsy samples were taken at time zero immediately after reperfusion. All results were normalised to one arbitrarily chosen sample.
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