Proper sampling of tissues is important in many ways for microarray studies. In conducting a hepatotoxicity study, capturing details of the histopathologic observations and correlating them with signature sequences identified in microarray studies allow an investigator to determine whether these parameters indeed represent the experimental conditions used. Standardization of all aspects of a microarray study, from tissue collection to data preparation for statistical analysis, reduces the variability among studies and increases an investigator's confidence in study results. The purpose of this study was to determine whether gene-expression differences exist between a random tissue cube used in a microarray study and a more homogenous pulverized, powdered sample.
Histopathologic evaluation revealed a variable hepatic response of centrilobular necrosis within the left lobe of some animals and among animals 24 hours following treatment with a toxic dose (1500 mg/kg) of APAP. That food was not removed from the animals prior to APAP dosing may in part be responsible for the varied degree of centrilobular necrosis; however, because not all animals respond in an analogous manner to toxicity testing, differences among tissue sampling methods for microarray studies must be identified (McLean et al., 1975
; Richardson et al., 1986
; Irwin et al., 2005
). Collecting liver sections adjacent to either side of the central lobe core used for genomic studies complements correlation of the experimental conditions between the histopathologic observations and microarray analysis.
Multiple statistical approaches failed to distinguish differences between the powdered and cubed sampling methods that increased variability would occur in the gene-intensity values from the random cubes compared to the homogenous powdered aliquots was speculated; however, this was not the case. Traditional bioinformatics tools used for categorizing groups, such as PCA and unsupervised hierarchial clustering proved unable to separate the 2 sampling methods. While major components of the variability did not differentiate the powdered from the cubed samples, minor components of the gene-expression variability may be attributed to sampling determined by PCA. Cluster analysis applied individually to the powdered and cubed samples also failed to categorize the sampling groups. While the more homogenous powdered group clustered for each animal, this occurrence was not observed in the cubed group. On the contrary, cubed samples from 2 animals (#5 and #18) did not cluster. In addition, these animals displayed moderate variability in their clinical-chemistry ALT values and extreme differences in their degree of centrilobular necrosis within the left lobe. Clearly, the cohesiveness of the cluster was disturbed by the random sampling method.
ANOVA analysis yielded a small number of signature sequences that were different between the powdered and cubed samples; however, when the same analysis was performed with a random assignment of arrays to the 2 groups, a similar result was achieved again, implying that no detectable difference could be discerned. This lack of difference was also the case for our statistical method, which evaluated the absolute deviation of each gene. Our screening method was designed to eliminate genes with high levels of variability, regardless of the sampling method. Removal of these genes with levels of increased variability should have made the differences in the variation between the sampling methods easier to detect if it was present but should not have created false differences between sampling methods when none existed.
A definitive reason could not be found to explain why there was no difference in gene expression, even though the degree of necrosis ranged from absent to marked with-in and between treated animals. Although necrotic hepatocytes were present in the sample, sufficient amounts of intact, non-degraded RNA were present to perform microarray. If the powdered sample had been from the remaining left lobe instead of the middle lobe core, separation between the two sample populations might have been more distinct.
Clear advantages and disadvantages exist between the powdered and cubed sampling methods. For all studies involving microarray, obtaining non-degraded RNA of the highest integrity constitutes the primary goal. A pulverized powder sample allows for multiple molecular procedures to be run from a more uniform sample, therefore minimizing study variability. Preparation of the pulverized powder sample is laborious, and great care must be taken to maintain a non-degraded RNA sample. Evaporation of LN2 in the powdered preparation must not occur and provisions ensuring sample integrity during the measurement and distribution of the powdered aliquot must be maintained; otherwise, the risk of degraded RNA becomes greatly increased. While isolating RNA from a cubed sample is simple and rapid if using a column-based assay from a commercial kit, two disadvantages are clear. First, a small, single representation of the target tissue is assayed to determine whether the total experiment is represented. If a commercial product is used for RNA preservation, the sample can be used solely for the molecular analysis of RNA. While, therefore, more labor is required and great care must be exercised in the preparation of a pulverized powder sample, the advantages of greater representation and the ability to investigate other downstream genomic applications (i.e., proteomics and metabonomics) outweigh the benefits of a random cubed sample.
Although this study did not detect conspicuous differences and establish a need for the utilization of one sampling method over another, we do advise our tissue collection method for histopathologic and microarray assessment of hepatocellular toxicity following treatment with a chemical compound. Collecting liver sections adjacent to either side of the central lobe core used for genomic studies allows improved correlation of the experimental conditions by histopathology and microarray. While the effect of the powdered samples in reducing variability is minimal, any improvements in the precision of assessing gene expression levels contribute to the potential identification of key toxicity markers following exposure to specific chemical compounds.