Testing an aflatoxin B1 gene signature in rat archival tissues - PubMed (original) (raw)
. 2012 May 21;25(5):1132-44.
doi: 10.1021/tx3000945. Epub 2012 May 4.
Affiliations
- PMID: 22545673
- PMCID: PMC3358548
- DOI: 10.1021/tx3000945
Testing an aflatoxin B1 gene signature in rat archival tissues
B Alex Merrick et al. Chem Res Toxicol. 2012.
Abstract
Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.
Conflict of interest statement
Notes: The authors declare no competing financial interests.
Figures
Figure 1. qPCR of AFB1 gene signature
Bar graph shows fold changes of aflatoxin B1 (AFB1)-induced gene expression in rat liver samples comparing qPCR data of formalin-fixed paraffin embedded (FFPE) liver RNA and fresh frozen (FF) liver RNA and microarray (MA) data published in Auerbach et al. Male F344 rats were exposed subchronically to AFB1 in feed at 1 ppm as described in Methods. DNA microarray analysis was performed with Agilent 4x44K rat whole genome oligonucleotide arrays. Based on microarray data, genes were selected for qPCR testing with FF liver and for comparative analysis with FFPE tissue blocks. Using the same animals from the microarray study, qPCR was performed with RNA extracted from either FF or FFPE samples. Bars represent means ± standard error of the mean (S.E.M.) at n=6 per group, which were compared by ANOVA and Tukey HSD post-hoc tests. For each gene, letters not shared among bars were different at p ≤ 0.05.
Figure 2. Regression analysis and principal component analysis (PCA) plots of liver gene expression data
Panel A. Regression analysis of AFB1-induced gene expression fold changes of transcripts analyzed by qPCR from fresh frozen (FF) or formalin-fixed paraffin embedded (FFPE) rat liver. Regression analysis was performed on fold changes for 2−ΔΔCt data for qPCR analysis of 14 genes from fresh frozen and FFPE liver samples at n=6 per group. A significant positive correlation at p ≤ 0.05 was determined for which an r2 value of 0.73 was obtained. Panel B. PCA shows the relationships among AFB1-induced liver transcript changes for each animal among microarray, FF and FFPE platforms. The plot accounts for 77.5% of variability in the data.
Figure 3. Molecular sizing of liver RNA and qPCR of AFB1-sensitive genes
Panel A shows representative microfluidic separations of RNA (900 ng/μl) isolated from formalin fixed, paraffin embedded (FFPE) liver of a control (C) or aflatoxin (AFB1) exposed rat while the insets are separations of fresh frozen (FF) liver RNA from the identical, respective animal. [FU] are fluorescent units; [nt] are number of nucleotides. Panel B shows overlays of amplification plots of cycle number vs fluorescence (ΔRn) for β-actin, Cdh13, Adam8 and Abcb1b transcripts for samples from control and AFB1 treatments. Each curve is a representative qPCR reaction from an individual control or AFB1 treated animal at n=6/group. Ct values for β-actin (mean ± SEM) were not significantly different between treatments so that their values were used for normalization of other transcripts. Relative fold changes from control were calculated by the 2−ΔΔCt method normalized against β-actin expression for each transcript as summarized in Figure 1.
Figure 4. Exon amplification of Akr7a3 transcript from cDNA of Fresh Frozen (FF) and Formalin Fixed Paraffin Embedded (FFPE) liver
Amplicons were from the 5′-untranslated region (5′-Utr) to Exons 1, 2, 3 and 7, and from Exon 1–2 or Exon 2 only. Results are representative of an experiment using fresh frozen and FFPE RNA from the same animal (AFB1). Three animals were evaluated and showed similar results. Note the uniform staining intensity of amplicons in fresh frozen liver while amplicons from paraffin samples (FFPE) liver were of decreased intensity with increasing product length, becoming faint at 499 bp and undetectable for the longest amplicon at 988 bp. Each PCR reaction was conducted for 35 cycles using primers with similar Tm values.
Figure 5. PCR cloning of coding regions for two AFB1-responsive genes
PCR primers were designed to form products that encompassed the coding region for each gene. Products were detected by UV light after separation on 2% agarose-ethidium bromide gels. For Akr7a3, the left panel shows 538 bp and 751 bp amplicons while the right panel contains Ddit4l amplicons of 296 bp and 513 bp sizes. cDNA was extracted from each band, cloned and Sanger sequenced. The identity of each band was confirmed by comparison to the mRNA sequence for Akr7a3 (NM_013215) and Ddit4l (NM_080399). Representative PCR reactions at 35 cycles are shown for control and AFB1 samples from fresh frozen liver.
Figure 6. qPCR of RNA isolated from kidney in paraffin blocks
Rats were exposed to AFB1 in feed for 90 days as described in Figure 1. Kidneys were formalin fixed, embedded in paraffin (FFPE), and after use, blocks were placed in archival storage. Subsequently, qPCR was performed after extraction of RNA from kidney in paraffin blocks. Data were normalized as the difference of Ct values for each gene and β-actin (ΔCt). Bars represent means ± standard error of the mean (S.E.M) at n=6 per group which were analyzed by ANOVA followed by comparison of control and AFB1 treatments using the Tukey HSD post-hoc test. For each gene, letters not shared among bars were different at p ≤ 0.05.
Figure 7. Molecular sizing and qPCR of liver, kidney and lung RNA
In Panel A, microcapillary separation of RNA was conducted to show the distribution of molecular weight sizes present in representative samples of liver, kidney and lung at 250 ng/μl. Fluorescent units [FU] and the number of nucleotides [nt] are on the ‘y’ and ‘x’ axis, respectively. Note flattening of fluorescence curve for high molecular sizes >4000 region in liver, while kidney and lung still show some fluorescence in this region (not RNA). In addition, note changes in shape of fluorescence-sizing curve of liver FFPE RNA in Figures 3a and 7a due to input differences in RNA concentration. Panel B presents qPCR data of constitutive genes, β-actin and S18, after RNA isolation from liver, kidney and lung in paraffin blocks. Bars represent means ± standard error of the mean (S.E.M) at n=6 per group which were analyzed by ANOVA and compared by Tukey HSD post-hoc tests. For each gene, letters not shared among bars were different at p ≤ 0.05.
Figure 8. Expression platforms for archival specimens
Gene expression based on protein, RNA and DNA analysis in fresh frozen tissues is often preferred for the greatest flexibility in biochemical and molecular analysis to complement conventional histopathology and immunohistochemistry (Histopath/IHC). However, several molecular platforms are compatible with formalin fixed paraffin embedded (FFPE) tissues for analysis of individual genes or genomic level studies. Platforms for FFPE RNA include qPCR (quantitative or real-time PCR), ISH (In Situ Hybridization) and qNPA (quantitative Nuclease Protection Assay), which can measure transcripts in single or multiplexed modes. Sub-genomic analysis can be conducted with oligonucleotide-based microarrays such as DASL (cDNA-mediated annealing, selection, extension, and ligation analysis) and bDNA (branched-chain DNA amplification for captured RNA) or genome-wide analysis by 3′-Seq (NextGen sequencing for FFPE adapted toward 3′-region of transcripts) or hybridization-based microarrays. Further, paraffin samples can be extracted for genomic or methylated DNA and be analyzed by sequencing or specialized microarray formats to link epigenetic effects (dotted arrow) with RNA expression.
References
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