Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach
© Pan et al; licensee BioMed Central Ltd. 2006
Received: 25 February 2006
Accepted: 31 May 2006
Published: 31 May 2006
The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes.
We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis.
The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies.
Microarray is a powerful technique for simultaneously determining the expression of thousands of genes [1–3]. Such studies can quickly yield a genome-wide description of mRNA expression, called transcriptomes, in a given cell or tissue at a given physiologic or pathologic condition . Even though, one of the main challenges in such genome-wide gene-expression profiling is the difficult inspection of genes with rare transcripts.
On the other hand, PCR-based suppressive subtractive hybridization (SSH) techniques are highly sensitive for identifying differences in gene expression [5–9]. However, the potentials of SSH in assaying dynamic changes of gene expression in minute levels have never been addressed before. In addition, SSH techniques are also restricted in terms of limited specificity and difficulties in identifying enriched genes. Combining the SSH technique with high-throughput screening of the harvested clones through the use of cDNA microarrays could greatly reduce the tedious work for northern blot analysis, as well as the likelihood of false-positive clones enriched via SSH . Such combined approaches by printing the clones obtained from the SSH amplicones on chips have been successfully used for profiling the differentiation of gene expression [10–13]. Nevertheless, in such approaches, the genes of the subtracted clones remain to be sequenced for identification, and a large portion of redundancy in the enriched amplicons must also be identified. Moreover, since the targets used to hybridize the amplicon clones printed on chips were the un-enriched cDNA pools rather than the subtracted, enriched clones, such approaches would not increase the sensitivity for detection of the low-expression genes.
Herein, we report our modifications using the subtracted amplicons as the targets to hybridize the pre-prepared microarray chips for SSH/microarray analysis. Since all of the probes on the microarray chips have been well characterized, the hundreds and thousands of genes in the subtracted amplicons can be determined by a single hybridization. Moreover, the relative expression status between the compared tissues for each gene can be augmented and easily determined by combining the use of the targets prepared both from forward and reverse subtractions of SSH. We named this modified approach "SSH/microarray" and used human hepatocellular carcinoma as a model to demonstrate the feasibility of this approach.
The SSH/cDNA microarray versus regular cDNA microarray approaches
The SSH/microarray approach on low-expression and differentially expressed genes
Since SSH specifically amplified the difference of gene expression, the SSH/microarray approach should also be able to identify more differentially expressed genes between hepatoma and non-hepatoma liver tissues, which were originally undetectable by the regular cDNA microarray methods. As shown in Fig 3C, of the 5015 low-expression genes identified only by the SSH/microarray assays, we identified additional 512 and 1923 genes with at least a 2-fold decrease and increase in hepatoma tissues, respectively.
Identification of differentially expressed genes with low expression in human hepatoma
Gene ontology analysis of the HCC differentially expressed genes
Gene ontology classification of the HCC differentially expressed genes
Regulation of apoptosis
Transcription & regulation of transcription
Cell organizaiton & biogenesis
Purine & pyrimidine metabolism
Ubiquitin ligase complex ©
Complement & coagulation cascades
Response to wounding
Response to external biotic stimuli
Cell ion homeostasis
Extra-cellular space ©
Microarray techniques are limited in the detection of genes with low expression , while subtractive hybridization methods are restricted by their specificity and the tremendous work needed for validating the results, as well as for sequencing to identify genes. There have been many reports using microarray techniques for rapid and high throughput validation of the subtraction specificity of SSH by spotting the enriched clones on the chips or membranes [10–13, 15–23]. However, such approaches would not only require tremendously sequencing works to identify genes including a great number of redundant clones in the subtracted amplicons, but also lose the sensitivity of SSH techniques to detect the low abundance transcripts, since the sensitivity of microarray analysis is determined by the targets used to hybridize chips but not by the probes printed on chips.
Herein we report our modified approaches. Instead of using the subtracted clones as probes spotted on chips, we directly labeled the enriched amplicons and used them as targets to hybridize the pre-made microarray chips for microarray analysis (named as "SSH/microarray" in this report). This approach allowed us not only to readily identify genes without tremendous sequencing works in the subtracted amplicons regardless of many redundant clones, but also to easily evaluate the subtraction efficiency and specificity. Moreover, the SSH/microarray approach made it possible to conduct a transcriptome-wide identification of differentially expressed genes particularly for those with low expression. It has been reported that the absolute expression level is not a crucial determinant for identifying genes, while the relative difference in expression levels does impact on whether or not a gene is recovered by subtractive hybridization [5, 24]. In this report using the SSH/microarray approach, we identified about ten times more of the differentially expressed genes, particularly for those with low expression, in human hepatoma. Our findings successfully demonstrated the transcriptome-wide assays of differentially expressed genes at low abundance. This simple but very powerful approach would greatly facilitate future researches on functional genomics [15, 19, 25].
One of the main concerns about SSH techniques is their specificity. However, as combined with microarray techniques, the subtraction efficiency can be readily evaluated. As shown in Figure 2, the subtraction efficiency was determined by the rare presence of clones with equal hybridization intensities between the forward and reverse amplicons, and by the consistent exclusion of the house-keeping genes in the subtracted amplicons. The specificity was further confirmed by quantification of the differential gene expression between hepatoma and para-hepatoma liver tissues using RT real-time PCR in the 9 randomly selected genes, of which six (GBA, PTPRF, GNL3, ERBB3, OGDH, FMO3) were identified to be differentially expressed only by the SSH/microarray assays, but not the conventional cDNA microarrays assays
Of interest, using this novel approach, we identified many of the genes potentially implicated in hepatocellular carcinogenesis, which were not previously identified by conventional cDNA microarray assays. For examples, 18 genes (WT1, NME2, IGF2, PPMIG, GMNN, PSMD2, CDK4, SFN, CDC20, CDC25C, AIF1, PINX1, CDC2, MSH2, PA2G4, ASPM, PAFAH1B1, DCTN3) implicated in cell cycle regulation, 16 genes (PLG, FABP3, APEG1, NCK1, RBBP4, NME2, CTBP1, FGA, IBFBP7, FGB, AIF1, CXCL1, JAG1, PINX1, GRN, PBEF1) correlated with the regulation of cell proliferation, 16 genes (HDAC1, DNASE1, NUDT2, CTNNAL1, HSAC1, BIRC1, FASTK, IGFBP3, TUBB, TNFSF10, TNFRSF11B, CLU, BFAR, BCAP31, BIRC2, C9) involved in the pathways of cell death, 35 genes (MYL4, APEG1, TTN, HTN1, MEST, EVL3, VLDLR, MYOG, TNNI2, HOXC11, IGF2, AHSG, TTID, BIRC1, PAFAH1B1, SYK, LMNA, TNFRSF11B, FALZ, BMP1, ETS2, NRD1, CXCL1, JAG1, SEMA4G, HEY1, ZNF22, CRMP1, TEAD4, IGFBP1, IGFBP3, IGFBP7, PTGS1, NEUGRIN, CUGBP1) related to the regulation of cell differentiation and embryonic development, and 59 genes (ASGR2, CAP2, RGS5, ITGB4BP, NCK1, EVL3, PLCG2, EPHA7, IGF2, IFNGR2, AHSG, CAP1, PTPRF, CXCL2, GNAI1, TGFBR1, NCSTN, DOK1, FRBB3, SYK, MC1R, CD79B, CSNKIE, CD69, AVPR1A, GNB2L1, ACVR1, BIRC2, FLT4, CXCL1, FAG1, PNOC, SLC9A3R1, LANCL1, GNB3, ADORA2B, FGR, NCK1, RAB11A, PLCG2, LOC91614, PRKAR1A, FLJ22595, ARF3, TYK2, MAP3K7IP1, MC1R, TNFSF10, ARF4, VAV2, AVPR1A, GNB2L1, MAPK10, STMN1, SNX17, ADORA2B, ECT2, RGS5, IGFBP3) associated with signal transduction in response to extra-cellular proliferation and growth stimuli were found. Our findings that differentially expressed genes were related to multi-biological processes suggest the complexity of the molecular mechanisms for hepatocellular carcinogenesis.
In this study, we modified the previously method of the combination of the suppressive subtractive hybridization and microarray techniques to differentially profile the low-expression transcriptomes of human hepatocellular carcinoma by directly labeling both of the reciprocal subtracted amplicons as target for cDNA microarray assays. Compared to SSH or previous SSH in conjunction with microarray approaches, this modified approach provided us with three additional advantages: 1) easy inspection of the subtraction efficiency, 2) avoidance of tremendous sequencing work for gene identification, 3) high sensitivity for identifying the low-expression, differentially expressed genes. This approach allowed for the detection of about ten times more of the differentially expressed genes than did the regular cDNA microarray approach in human hepatoma, particularly for those with low expression. Using this approach, we identified many genes potentially implicated in human hepatocarcinogenesis, which were not identified before. For its high efficiency and high sensitivity, this SSH/microarray approach is powerful for the rapid differentially profiling the low-expression transcriptomes, and most adequate for applying to functional genomic studies.
Tissues and patients
For SSH and cDNA microarray analysis, hepatoma and the corresponding non-cancerous liver tissues were obtained from 3 patients who had liver surgery at the Chang Gung Memorial Hospital. For reverse-transcription real-time PCR, hepatoma and the matched non-hepatoma liver tissues were obtained from additional 18 patients of hepatoma. Diagnoses of HCC and non-hepatoma liver tissues were based on histo-pathologic findings. The Internal Review Board for Medical Ethics of Chang Gung Memorial Hospital approved the specimen collection procedures and informed consent was obtained from each subject or subject's family.
Suppressive subtractive hybridization
Total RNA from hepatoma and the para-hepatoma liver tissues and the poly(A) RNA was prepared as described before [26, 27]. SSH was performed with the Clontech PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA) as described by the manufacturer but with the following modifications. Starting material consisted of 2 μg hepatoma mRNA as tester and 2 μg non-hepatoma liver tissue mRNA as driver, and vice versa. Primary and secondary PCR conditions were altered to increase specificity of amplification according to either plan A or B. Both A and B reduced the extension time and the number of cycles of the primary PCR to 2 min and 26 cycles, respectively. The primary PCR products were diluted 1/50 prior to use in the secondary PCR. All other aspects of plan A were as per the instructions of the manufacturer. Plan B diverged from plan A in two ways. First, the initial cycle of primary PCR was performed using annealing and extension times that had been reduced to 15 s and 1.5 min, respectively. Second, for subsequent cycles, the denaturing time was increased to 10 s while the annealing and extension times were reduced to 15 s and 1.5 min, respectively.
RNA labeling and microarray procedures
In this study, we used the GMRCL Human 15 K set, Version 2 chips as previously described , which contained 14,811 sequence-verified, human cDNA clones mapped to 12,530 distinct genes. All of the samples for the regular cDNA microarray or SSH/microarray assays were performed with the dye-swapping microarray design for minimizing labeling bias and statistical variances of data. For the regular cDNA microarray experiment, we used 2 μg of the total RNA for labeling and hybridization using a 3DNA Array 350RP Detection kit (Genisphere, PA, USA).
For the SSH experiment, 1 μl subtractive PCR products were labeled with Cy3 and Cy 5-dCTP (NEN, Boston, MA) using random primers. Unincorporated fluorescent nucleotides were removed using a Qiaquick PCR purification kit (Qiagen). The fluorescent-labeled DNAs were mixed with 30 μg of human cot-1 DNA (Invitrogen) and 100 μg yeast tRNA, precipitated and then resuspended in 30 μl of Microarray Hybridization Buffer Version 2 (Amersham Pharmacia). The hybridization solution was heated to 80°C for 10 min to denature the DNA and was then incubated for 30 min at 37°C, allowing cot-1DNA and yeast tRNA to block the repetitive sequences in genome probes. The probes were hybridized to a human cDNA microarray (GMRCL Human 15 K). We scanned the slides with a confocal scanner ChipReader (Virtek, Canada) and acquired the spot and background intensities with the GenePix Pro 4.1 software (Axon Instruments, Inc., CA, USA). The within-slide normalization was done using programs written with MATLAB 6.5 software (The MathWorks, Inc., MA, USA).
Reverse-transcription real-time PCR
To validate the results obtained from the SSH/microarray assays, we randomly selected six differentially expressed genes identified only by the SSH/microarray assays and three differentially expressed genes identified by both the regular cDNA microarray and SSH/microarray assays for the comparison of gene expression between hepatoma and the corresponding non-hepatoma liver tissues in eighteen patients of HCC using reverse transcription real-time PCR (qRT-PCR). Total RNA was extracted from tissues with Trizol reagents and reverse transcribed using the SuperScript III first strand synthesis system (Invitrogen, Carlsbad, CA). qRT-PCR was conducted using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Pre-designed Assays on Demand TaqMan probes and primer pairs for these 9 genes were obtained from Applied Biosystems Incoporated (ABI) (Foster City, CA). For each gene, two to four sets of Taq-Man probes and primers were tested. The probes contained a 6-carboxy-fluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and non-fluorescent quencher at the 3' end. These were designed to hybridize across exon junctions. As a result, no fluorescent signal was generated by these assays when genomic DNA was used as a substrate, which confirmed that the assays measured only mRNA. Equal amounts of RNA were used for all qRT-PCR reactions, which were performed in triplicate, and 18S ribosomal RNAs were used as internal controls.
Gene ontology analysis
All data (derived from three pairs of HCC and non-HCC liver tissues in a dye-swapping approach) were filtered so that 446 up-regulated and 255 down-regulated genes in HCC with at least two-fold difference and p value less than 0.01 were included in the further studies. After removal of the un-annotated genes, a total of 360 and 202 HCC up- and down-regulated genes were subjected to the subsequent gene ontology analyses. The two lists of the differentially expressed genes were analyzed using the on-line software FatiGo for comparative gene ontology categories including molecular function, biological process and cellular component . They were also imported into the on-line software KEGG2 for pathway mapping [30, 31]. The statistical significance was defined as P < 0.01 between the HCC up- and down-regulated gene groups using Fisher's exact test.
suppressive subtractive hybridization
This work was supported by a research grant from Chang Gung Memorial Hospital (CMRPG), Tao-Yuan, Taiwan.
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