RNAlater Storage Research Articles

DNA/RNA Preservation in Glacial Snow and Ice Samples.

The preservation of nucleic acids for high-throughput sequencing is an ongoing challenge for field scientists. In particular, samples that are low biomass, or that have to be collected and preserved in logistically challenging environments (such as remote sites or during long sampling campaigns) can pose exceptional difficulties. With this work, we compare and assess the effectiveness of three preservation methods for DNA and RNA extracted from microbial communities of glacial snow and ice samples. Snow and ice samples were melted and filtered upon collection in Iceland, and filters were preserved using: (i) liquid nitrogen flash freezing, (ii) storage in RNAlater, or (iii) storage in Zymo DNA/RNA Shield. Comparative statistics covering nucleic acid recovery, sequencing library preparation, genome assembly, and taxonomic diversity were used to determine best practices for the preservation of DNA and RNA samples from these environments. Our results reveal that microbial community composition based on DNA was comparable at the class level across preservation types. Based on extracted RNA, the taxonomic composition of the active community was primarily driven by the filtered sample volume (i.e., biomass content). In low biomass samples (where <200 ml of sample volume was filtered) the taxonomic and functional signatures trend toward the composition of the control samples, while in samples where a larger volume (more biomass) was filtered our data showed comparable results independent of preservation type. Based on all comparisons our data suggests that flash freezing of filters containing low biomass is the preferred method for preserving DNA and RNA (notwithstanding the difficulties of accessing liquid nitrogen in remote glacial field sites). Generally, RNAlater and Zymo DNA/RNA Shield solutions work comparably well, especially for DNA from high biomass samples, but Zymo DNA/RNA Shield is favored due to its higher yield of preserved RNA. Biomass quantity from snow and ice samples appears to be the most important factor in regards to the collection and preservation of samples from glacial environments.

Development of a Hadal Microbial In Situ Multi-Stage Filtering and Preserving Device

The unique environment of the hadal zone has created material circulation patterns and biological gene characteristics. Microbes play an irreplaceable role in the ocean ecological environment and material circulation due to their pervasiveness, abundance, and metabolic diversity. In this paper, we designed and developed a microbial sampling device that can be used in a depth of 10,000 m, with its working parts suitable for the full-sea depth. The multi-stage membrane realized the in situ multi-stage filtrations. The samples were in situ fixedly preserved by RNAlater storage solution. At the same time, we modeled and calculated the multi-stage membrane separation and filtration process, simulated the interception phenomenon of particles with different sizes passing through the multi-stage membrane area, and explored the influence of varying inlet velocities. A multi-stage membrane separation and filtration test system was built. The operational characteristics of different filters were compared and analyzed, and the appropriate filter material was selected according to the flow capacity and physical properties. A 100 MPa high-pressure test was carried out to check the device’s performance under a high-pressure environment. The sampler prototype was constructed and tested in the Mariana Trench. The results indicated that the device could work at the deepest point of the Mariana trench.

Processing and Analysis of Tissue Samples from Esophageal Cancer Patients in an African Setting.

Although infectious diseases continue to present a major health care problem in Africa, the incidence of cancer is increasing rapidly on the African continent and this merits an increased investment in cancer research in low to medium resource settings. Esophageal squamous cell carcinoma (ESCC) has a high incidence in Eastern and Southern Africa, with late clinical presentation and a very poor prognosis. There is limited research on the molecular pathology of this cancer in Africa, partly as a result of a lack of infrastructure for biobanking and sample processing in many African countries. The aim of this study was to establish a practical and robust workflow to collect, store, and process esophageal cancer samples such that both the tissue architecture and quality of the samples would be preserved and suitable for future genomic research. We developed a workflow that allows storage of fresh biopsy tissue in sterile Eppendorf tubes containing RNAlater, an efficient RNAse inhibitor. We collected 142 ESCC biopsy samples and showed that storage in RNAlater for up to 18 months did not alter tissue morphology, thus allowing histologic assessment by experienced pathologists and determination of tumor content in each biopsied sample. DNA and RNA extracted from tissue samples was assessed for purity, molecular size, and yield. The quantity and quality of nucleic acids obtained were suitable for genomic applications, and whole-exome sequencing of DNA from tumor tissues produced sequence data with a high proportion of both usable reads and correct base calling. We conclude that this workflow may be applicable to a wide range of malignancies for future genomic research in low-resource settings.

Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers

For proteome analyses, the tissue samples are mostly preserved either snap frozen or formalin-fixed, paraffin-embedded form. Use of RNAlater-a non-toxic solution primarily used to stabilize the RNA content of samples-in tissue preservation for proteome analysis recently described equally reliable with snap-frozen preservation in human tissues. Even though RNALater storage has great potential in the preservation of Peripheral Blood Mononuclear Cells (PBMC), its impact on the results of proteome analysis is poorly described at qualitative and quantitative measures. The present study investigated protein profiles of RNAlater preserved and fresh PBMCs using three extraction buffers viz. Triton X-100, RIPA and SDS. Proteins are separated in SDS-PAGE and quantified using densitometry. On an average 19.3 bands from fresh and 15.6 bands from RNAlater storage cells were obtained with a molecular weight ranging from 25 to > 250 kDa. RNAlater storage generated a fewer number and lesser quantity of low molecular weight proteins while yielded a similar or high quantity of high molecular weight protein fractions. The principal component analysis showed that Triton X-100 is inferior as compared to SDS and RIPA with respect to their protein bands and quantity yielded. While RNAlater is effective in preserving PBMC for proteome analysis, our findings warrant caution in its use in proteomics experiments especially if the target is low molecular weight proteins.

Comparison of the efficiency of different cell lysis methods and different commercial methods for RNA extraction from Candida albicans stored in RNAlater

BackgroundObtaining sufficient RNA yield and quality for comprehensive transcriptomic studies is cumbersome for clinical samples in which RNA from the pathogen is present in low numbers relative to the nucleic acids from the host, especially for pathogens, such as yeasts, with a solid cell wall. Therefore, yeast cell lysis including cell wall disruption constitutes an essential first step to maximize RNA yield. Moreover, during the last years, different methods for RNA extraction from yeasts have been developed, ranging from classic hot phenol methods to commercially available specific kits. They offer different RNA yield and quality, also depending on the original storage medium, such as RNAlater.ResultsWe observed that, for C. albicans cells stored in Tryptic Soy Broth with 15% glycerol, 10 min of bead beating in a horizontal position in RiboPure Lysis Buffer provided complete cell lysis. Cell lysis efficiency was decreased to 73.5% when cells were stored in RNAlater. In addition, the RiboPure Yeast Kit (Ambion) offered the highest RNA yield in comparison with the automated platform NucliSENS easyMAG total nucleic extraction (bioMérieux) and the RNeasy Mini Kit (Qiagen) according to NanoDrop and Fragment Analyzer. Moreover, we showed that, in spite of the decrease of cell lysis efficiency after RNAlater storage, as compared to storage in TSB + 15% glycerol, RNAlater increased RNA yield during RNA extraction with both RiboPure Yeast Kit and easyMAG, as confirmed by Fragment Analyzer analysis and by RT-qPCR of the RNA from the Internal Transcribed Spacer 2.ConclusionsIn our hands, the most efficient cell lysis and highest RNA yield from C. albicans cells stored in RNAlater was obtained by horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the RiboPure Yeast Kit.

Expression Differences of Stress and Immunity Genes in Rainbow Trout (Oncorhynchus mykiss, Walbaum 1792) with Different Bacterial Fish Diseases

The aim of this study was to determine the changes in the mRNA transcription levels of HSP70 and IGF genes related to stress and immunity in Oncorhynchus mykiss obtained from fish farms, to determine the phenotypic and antimicrobial properties of the bacteria isolated in the study and to compare the results according to different diseases. Accordingly, six fish from each fish farm, a total of 30 fish were sampled. Bacterial identification, inoculations were carried out from the tissue samples taken from the kidney and symptomatic surfaces of fish samples in TSA. Primary cultures were obtained after incubation periods at 21 and 37oC. Gram staining, catalase, and oxidase tests were performed. Forty-six biochemical and 26 antimicrobial tests were performed using BD Phoenix ID Panels. Muscle tissue was used to determine mRNA gene expression differences and the tissues were preserved at -80°C in RNAlater storage solution. Following the RNA isolation and cDNA synthesis, a real-time PCR procedure was performed with b-Actin (ACTB), Insuline-like growth factor (IGF) and HSP70 Gene Primer Array system. Enterococcus faecium (E. faecium), Lactococcus garvieae (L. garvieae) and Staphylococcus aureus (S. aureus) agents were isolated in the study. These bacteria were identified with 91-99% similarity ratios. No disease agents in the gene expressions of the fish samples in were isolated and they were therefore used as controls. Compared to the control group, heat shock protein (HSP) mRNA expression was upregulated in fish tissues infected with E. faecium, S. aureus, and L. garvieae whereas no significant increase was determined in fish tissues infected with S. aureus. IGF mRNA gene expression was upregulated in all infected tissues. IGF expression was upregulated at the highest level in fish tissues infected with L. garvieae.

Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols

BackgroundThe human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly.MethodsFecal samples from healthy adults were stored at -80°C as such or diluted in RNAlater® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance.ResultsWe obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon.ConclusionOur results showed that RNAlater® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.

Fish viruses stored in RNAlater can remain infectious and even be temporarily protected from inactivation by heat or by tissue homogenates

RNAlater is a commonly used transport and storage solution for samples collected for fish health investigations, particularly those potentially involving viruses. However, the infectivity of fish viruses after storage in RNAlater have not been determined. Nevertheless, knowledge of pathogen infectivity of preserved samples is crucial for ensuring safe transport and storage protocols. Therefore, the infectivity of three fish RNA viruses in RNAlater was examined at four temperatures: −80 °C, 4 °C, room temperature (RT, approximately 22 °C) and 37 °C. The viruses were viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV) and chum salmon reovirus (CSV). Overall, three consistent outcomes were observed. First, all three viruses remained infectious in RNAlater at RT or lower. High log titres of these viruses remained over 30 d of storage in either RNAlater or PBS. Second, RNAlater delayed the thermal inactivation of these viruses when compared to PBS at 37 °C. For VHSV, the titre remained high in RNAlater after one day of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For IPNV, the titre remained high in RNAlater after 30 d of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For CSV, the titre was slightly higher in RNAlater than PBS at 37 °C over 7 d, and by day 30, only samples stored in RNAlater proved infectious at titres above the detection threshold. Third, RNAlater delayed the inactivation of these viruses when they were stored together with head kidney homogenates. For VHSV, infectious virus was recovered from samples stored at 4 °C in RNAlater by day 7 of incubation, whereas it was inactivated to below threshold in PBS over the same period. For both IPNV and CSV, infectious virus was recovered from samples stored at 37 °C in RNAlater for 7 d, but not so in PBS. In summary, fish viruses can remain infectious and are even temporarily protected from inactivation while in RNAlater. This makes RNAlater a potentially useful solution for the transport of fish viruses. At the same time, precautionary measures must be taken when transporting potentially infectious samples in RNAlater.

Common methods for fecal sample storage in field studies yield consistent signatures of individual identity in microbiome sequencing data.

Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples—unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. Here, we extend previous work on storage methods for gut microbiome samples by comparing immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: lyophilization, storage in ethanol, and storage in RNAlater. We found that the signature of individual identity consistently outweighed storage effects: alpha diversity and beta diversity measures were significantly correlated across methods, and while samples often clustered by donor, they never clustered by storage method. Provided that all analyzed samples are stored the same way, banked fecal samples therefore appear highly suitable for investigating variation in gut microbiota. Our results open the door to a much-expanded perspective on variation in the gut microbiome across species and ecological contexts.

Abstract 4010: Prognostic role of miRNAs in resectable non-small cell lung cancer

Abstract BACKGROUND MicroRNAs (miRNA) are a class of small RNAs that regulate gene expression, and are implicated in several diseases including cancer. The aim of this study was to analyze the prognostic implications of deregulated miRNAs identified by NGS in a previous study performed on resectable-NSCLC tumor samples. MATERIAL AND METHODS Fresh-frozen tumor and normal adjacent lung tissue samples (N = 195) were obtained by surgical resection from NSCLC patients and storage in RNAlater® at -80°C until analysis. RNA was isolated biological specimens by Trizol based-methods. 22 deregulated miRNA were analyzed in paired tumor/normal samples by RTqPCR using TaqMan® microRNA assays. Statistical analyses were considered significant at p&amp;lt;0.05. RESULTS Patient's median age was 64 years [26-85], 88.2% were male, 66.7% had good performance status (PS = 0), 39.5% were adenocarcinomas and 54.9% presented stage I at the time of the diagnosis. For survival analysis, continuous variables were dichotomized as high (≥ median) and low (&amp;lt; median), using the median expression of each miRNA as a cutoff. Of the 22 miRNAs analyzed only miR-21 and miR-188-5p had prognostic value. We found that patients with high levels of miR-21 and miR-188-5p had a significant reduction in PFS and OS rates. Interestingly, the group of patients with the combined high expression of both miRNAs (miR-21high &amp; miR-188-5phigh) showed shorter PFS and OS (Table 1). Clinico-pathological and analytical variables were included in the multivariate analysis revealing that combined miRNAs is an independent prognostic marker for PFS (HR 0.485 [0.313-0.753]; p = 0.001) and OS (HR 0.389 [0.237-0.638]; p&amp;lt;0.0001]. a P value with Bonferroni correction are shown in parenthesesOSOSPFSPFSMedian (months)PaMedian (months)PamiR-21 (High vs low)42.60 vs 82.600.01622.13 vs 66.970.017miR-188-5p (High vs low)46.67 vs NR0.00524.03 vs 66.970.023Combined miRNAs (miR21 high miR-188 high vs combonations)21.13 vs 83.570.002 (0.008)16.97 vs 56.830.001 (0.004) CONCLUSION Expression of miR-21 and miR-188-5p seems to have a prognostic role in NSCLC, and the combination of both could be an independent prognostic marker in early-stage NSCLC. Table 1. OS and PFS for miR-21 and miR-188-5p in NSCLC patients (univariate analysis) Supported by grants RTICC (RD12/0036/0025 and RD06/0020/1024), TRACE (TRA09-0132) and Beca Roche Oncohematología. Citation Format: Sandra Gallach, Eloisa Jantus-Lewintre, Silvia Calabuig-Fariariñas, Ana Blasco, Ricardo Guijarro, Miguel Martorell, Carlos Camps. Prognostic role of miRNAs in resectable non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4010. doi:10.1158/1538-7445.AM2015-4010

Evaluation of different storage methods to characterize the fecal bacterial communities of captive western lowland gorillas (Gorilla gorilla gorilla)

Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.

A field method to store samples from temperate mountain grassland soils for analysis of phospholipid fatty acids

The storage of soil samples for PLFA analysis can lead to shifts in the microbial community composition. We show here that conserving samples in RNAlater, which is already widely used to store samples for DNA and RNA analysis, proved to be as sufficient as freezing at −20 °C and preferable over storage at 4 °C for temperate mountain grassland soil. The total amount of extracted PLFAs was not changed by any storage treatment. Storage at 4 °C led to an alteration of seven out of thirty individual biomarkers, while freezing and storage in RNAlater caused changes in the amount of fungal biomarkers but had no effect on any other microbial group. We therefore suggest that RNAlater could be used to preserve soil samples for PLFA analysis when immediate extraction or freezing of samples is not possible, for example during sampling campaigns in remote areas or during transport and shipping.

RNA Stability in Human Liver: Comparison of Different Processing Times, Temperatures and Methods

The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1α and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank.

Impact of Collection and Storage of Lung Tumor Tissue on Whole Genome Expression Profiling

Gene expression profiling could assist in revealing biomarkers of lung cancer prognosis and progression. The handling of biological samples may strongly influence global gene expression, a fact that has not been addressed in many studies. We sought to investigate the changes in gene expression that may occur as a result of sample processing time and conditions. Using Illumina Human WG-6 arrays, we quantified gene expression in lung carcinoma samples from six patients obtained at chest opening before and immediately after lung resection with storage in RNAlater [T1a((CO)) and T1b((LR))], after receipt of the sample for histopathology, placed in RNAlater [T2a((HP))]; snap frozen [T2b((HP.SF))]; or snap frozen and stored for 1 week [T2c((HP.SFA))], as well as formalin-fixed, paraffin-embedded (FFPE) block samples. Sampling immediately after resection closely represented the tissue obtained in situ, with only 1% of genes differing more than twofold [T1a((CO)) versus T1b((LR))]. Delaying tissue harvest for an average of 30 minutes from the operating theater had a significant impact on gene expression, with approximately 25% of genes differing between T1a((CO)) and T2a((HP)). Many genes previously identified as lung cancer biomarkers were altered during this period. Examination of FFPE specimens showed minimal correlation with fresh samples. This study shows that tissue collection immediately after lung resection with conservation in RNAlater is an optimal strategy for gene expression profiling.

Optimization of RNA Preservation in Granulation Tissue in the Expanded Polytetrafluoroethylene (ePTFE) Tube Model

RNA is easily degraded by RNAses. An efficient and non-hazardous method optimizing RNA preservation and extraction is needed. Our aim was to evaluate RNA preservation methods on murine granulation tissue from subcutaneously implanted ePTFE tubes. ePTFE tubes were placed subcutaneously in mice, removed after ten days and randomized to four RNA preservation groups. Storage in RNAlater at 4°C and snap freezing in methanol/acetone yielded significantly better RNA quality than snap freezing with liquid nitrogen and storage in RNAlater at −80°C. Snap freezing in liquid nitrogen failed to yield intact RNA. We recommend storage in RNAlater at 4°C.

Optimization of a protocol for extraction of Plasmodium falciparum RNA from infected whole blood samples for use in DNA microarrays

This study was carried out to determine the efficiency of two reagents, RNAlater and RNAwiz, for their ability to stabilize Plasmodium falciparum RNA in infected whole blood and saponin lysed parasite pellets for use in DNA microarrays. Eight infected blood samples were stored in each of the reagents, and RNA extracted at days 0 and 56 post collection. RNA yields and quality were compared at the different time points between the two test reagents. We show that for both reagents, higher RNA yields and quality is obtained when RNA is isolated immediately after sample collection (day 0), however, results show that RNAwiz storage provides a marginally higher RNA yield compared to RNAlater storage. Our results indicate that whole blood gave slightly higher RNA yields with superior quality as compared to saponin lysed samples when such whole blood samples are stored in RNA wiz, but not in RNAlater. From our results, we recommend RNAwiz as a better reagent for use in storage of whole infected blood intended for extraction of P. falciparum RNA for DNA microarrays and other sensitive techniques.

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNAlater at 4 °C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.