Laser Capture Microdissection Technique Research Articles

Laser microdissection-based expression analysis of key genes involved in muscle regeneration in mdx mice

We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis–regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin–Eosin staining, different types of degenerative–regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3–5 days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFβ-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5–7 days post-necrosis). mdx degenerative–regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.

EXPRESSION ANALYSIS OF TRANSCRIPTS FOR GERM CELL-SPECIFIC GLYCOLYTIC ENZYMES (HK1S, GAPDHS, PGK2, AND LDHC) USING LASER CAPTURE MICRODISSECTION

Hexokinase is the first enzyme in the pathway of glycolysis. Previous studies identified three spermatogenic cell-specific variant transcripts for type 1 hexokinase; Hk1_v1, Hk1_v2 and Hk1_v3 (formerly named Hk1-sa, Hk1-sb, and Hk1-sc). They encode a common protein, HK1S, found mainly in the principal piece region of the sperm flagellum. Subsequent studies measured the steady-state level of the variant transcripts by real time RTPCR (qPCR) and demonstrated that they correlate with the expression of these transcripts in spermatogenic cells isolated from sections using laser capture microdissection (LCM). In the present study, we used LCM and qPCR to define the patterns of expression of transcripts for other spermatogenic cell-specific glycolytic enzymes that are localized to the principal piece region of the sperm flagellum. Cryostat sections of testes from adult mice were fixed in cold acetic acid:ethanol (1:19) and used for the isolation of spermatogonia, pachytene spermatocytes, early spermatids and late spermatids (4000 cells/each cell type, 3 replicates) by LCM. The purity of the isolated spermatogonia and pachytene spermatocytes was determined by RT-PCR analysis using primers for cell-specific transcripts. The steady-state levels of the three spermatogenic cellspecific Hk1 variant transcripts and of three other spermatogenic cellspecific glycolytic enzymes were determined in these cell types using qPCR analysis. The qPCR analysis revealed that the steady-state levels of Hk1_v2 and Hk1_v3 transcripts were highest in late spermatids. Glyceraldehyde 3-phosphate dehydrogenase (Gapdhs) and phosphoglycerate kinase 2 (Pgk2) steady-state transcript levels also were highest in late spermatids. However, the steady-state levels of Hk1_v1 and lactate dehydrogenase C (Ldhc) transcripts were lower in late than in early spermatids. Thus, transcripts for these germ cell specific glycolytic enzymes have two patterns of expression in spermatogenesis. It remains to be determined if transcripts for other glycolytic enzymes present in sperm are expressed in one of these two patterns. Our present results suggest that LCM technique is useful for investigating the steady-state level of germ cell-specific transcripts throughout spermatogenesis. These studies were supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. (poster)

Age-related down-regulation of HCN channels in rat sinoatrial node

Aging is associated with deteriorated sinoatrial (SA) node function. The pacemaker current (If) carried by hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels plays a key role in the generation of spontaneous activity of the SA node cells. In the present study, the SA node cells were identified and isolated using the laser capture microdissection (LCM) technique for quantitative analysis of the HCN channel isoforms HCN1-HCN4 transcripts. Using real-time quantitative reverse transcription- polymerase chain reaction (RT-PCR), marked down-regulated transcriptions of HCN2 and HCN4 were observed in the SA node from young (1-month-old) to adult (4-month-old) and further to aged (30- month-old) rats. However, neither the HCN1 nor HCN3 transcript was detectable throughout the lifespan of the rat. Consistently, the effect of 2 mM Cs+ to selectively block the HCN channels, on pacemaking was also lessened with age. Our findings raise the possibility that the down-regulated transcription and relative function of HCN channels may contribute to the decline of the SA node function in aged rats.

Tissue integrity and RNA quality of laser microdissected phloem of potato

The phloem is an important conduit for the transport of signaling molecules including RNA. Phloem sap has served as a source of RNA to profile uncontaminated phloem transcriptomes but its collection is difficult in many species. Laser capture microdissection techniques offer a valuable alternative for isolating RNA from specific vascular cells. In potato (Solanum tuberosum L.), there are seven BEL1-like transcription factors expressed throughout the plant with diverse functions. The RNA of one of these, StBEL5, moves through the phloem from the leaf to stolon tips to regulate tuber formation. In this study, the presence of several BEL RNAs and one Knotted1-like RNA was determined in phloem cells collected by laser microdissection coupled to laser pressure catapulting (LMPC). Three fixatives were compared for their effect on cell morphology and RNA quality in transverse sections of stems of potato. For optimum tissue integrity and quality of RNA from potato stem sections, the best results were achieved using ethanol acetate as the fixative. In addition, the RT-PCR results demonstrated the presence of six out of seven of the StBEL RNAs and a potato Knox RNA in phloem cells.

Fibre type distribution and gene expression levels of both succinate dehydrogenase and peroxisome proliferator‐activated receptor‐γ coactivator‐1α of fibres in the soleus muscle of Zucker diabetic fatty rats

We have reported that a change in muscle fibre type distribution is present in two strains of diabetic rats (Otsuka Long-Evans Tokushima Fatty and Goto-Kakizaki rats). In this study, we determined whether the change in soleus muscle fibre type distribution was caused by diabetes, using obese, diabetic (Zucker diabetic fatty, ZDF), obese, non-diabetic (Zucker fatty, ZF) and non-diabetic, non-obese rats (Zucker lean, ZL). Moreover, we investigated whether the gene expression levels of metabolic key molecules, namely the transcriptional factors of metabolic genes, exemplified by peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), and the oxidative enzymes in mitochondria, exemplified by succinate dehydrogenase (SDH), were changed in type I and II muscle fibres in each type of rat, using the new technique of laser capture microdissection (LCM). Both plasma glucose and glucosylated haemoglobin levels were significantly higher in ZDF than in ZL and ZF rats. A lower percentage of type IIA fibres was observed in the muscles of ZDF rats than in those of ZL and ZF rats. The mRNA expression levels of SDH in type II fibres and of PGC-1alpha in type I fibres were significantly lower in ZDF than in ZL and ZF rats as assessed by LCM and real-time PCR analysis. We have shown, for the first time, that a lower percentage of type IIA fibres was observed in ZDF rats. We have also discovered that the expression levels of the oxidative metabolism-related genes, PGC-1alpha and SDH, decreased in type I and type II fibres, respectively, of ZDF rats.

The TP53 and RPS6 alterations at the invasive tumor front, center and stroma of oral squamous cell carcinoma

To assess the difference of genetic alteration patterns among different areas in the same oral squamous cell carcinoma (OSCC). Studied the loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosomal loci TP53 and RPS6 on the invasive tumor front (ITF), the center/superficial part and stroma cells by combining laser capture microdissection (LCM) and PCR technique. There existed a high frequency of LOH and MI on chromosomes loci TP53 and RPS6. The frequency of RPS6 and TP53 aberration at the stroma was 23.5% (4/17) and 43.8% (7/16), respectively. While in epithelial part (both ITF and center), it reached up to 64.7% (11/17) and 70.6% (12/17) respectively, and the difference was significant (P < 0.05). The overall frequency of the two markers was statistically higher at the ITF (20/32) than at the center/superficial part (15/34) (P < 0.05). The current study revealed that genetic alterations were different in different areas of the same tumor and there existed a relationship between the histological grading and genotypes of OSCC.

Isolation of outer hair cells from the cochlear sensory epithelium in whole-mount preparation using laser capture microdissection

Outer hair cells (OHCs) play an important role in frequency selectivity and signal amplification in the mammalian cochlea. Because OHCs are relatively few in number and a minority of the cells in the cochlea, separating and isolating them for applications such as cDNA library creation and proteomic studies is a challenging task. Laser capture microdissection (LCM) is designed to capture cells from very thin tissue sections, it can accurately isolate specific cells from large regions of tissue for RNA, DNA, and proteomic studies. Due to the constraints of cochlear anatomy, thin sections of the cochlea contain small numbers of OHCs. Therefore, we adapted the LCM technique to isolate OHCs from organ of Corti whole-mounts, each of which contain hundreds of OHCs that are simultaneously accessible and collectable. For comparison, we also used a more traditional mechanical dissection. The quality of cDNA derived from the OHCs collected with LCM and with the traditional mechanical method are compared and the merits and limitations of the techniques discussed. A similar approach can also be used to isolate large quantities of inner hair cells and selected supporting cells from the whole-mount cochlear preparation.

Application of laser capture microdissection and differential display technique for screening of pathogenic genes involved in endometrial carcinoma

The objective is to eliminate the interference from other cell types; gene fragments involved in endometrial carcinoma (EC) are screened and cloned. Human normal endometrial glandular epithelia and EC cells were harvested with laser capture microdissection (LCM). The purification and concentration of minimal RNA were used to screen differential expressed gene fragments involved in EC by fluoro differential display polymerase chain reaction (FDD-PCR). The differential gene fragments were cloned, sequenced, and then identified by reverse Northern blot hybridization. Positive fragments were analyzed with basic local alignment search tool (BLAST). Cyclin-dependent kinase 7 (CDK7) expressions in EC and normal endometrial tissue were tested by immunohistochemical staining. Of 38 differential bands, 3 bands were of high expression in normal endometrium and 35 in EC. Six positive differential gene fragments were obtained and BLAST analysis for them suggested that L1.1 was homologous (99% identical) to the CDK7; L1.9 had a 99% homology with protein phosphatase 1 regulatory (inhibitor) subunit 12 A (PPP1R12A); L1.21 and L1.22 showed a 100% homology with cellular repressor of E1A-stimulated genes 1 (CREG); and L1.25 and L1.26 indicated more than 98% homology with solute carrier family 39 (zinc transporter), member 10 (SLC39A10). Immunohistochemistry revealed that CDK7 expression was higher in EC than in normal endometrium. We conclude that pathogenic genes involved in EC are obtained with LCM and FDD-PCR. It has been first found that CDK7, PPP1R12A, CREG, and SLC39A10 are correlative with EC from gene level. CDK7 is strongly associated with EC and can be used as potential molecular marker of EC for further studies.

Analysis of glomerular VEGF mRNA and protein expression in murine mesangioproliferative glomerulonephritis

Capillary repair is crucial in the healing of glomerulonephritis (GN). The vascular endothelial growth factor (VEGF) has pro-angiogenic properties and plays an important role in glomerular capillary regeneration. Habu Snake Venom (HSV) GN, a murine model for mesangioproliferative GN, was induced in uninephrectomized C57/BL6 mice. Glomerular damage and capillary repair were assessed using morphometry, stereology, and confocal laser scanning microscopy. Mesangiolytic glomeruli were microdissected (days 1,3,7,14) using laser capture microdissection technique. VEGF mRNA expression was analyzed by real-time polymerase chain reaction and compared to intact glomeruli of healthy controls. Spatiotemporal VEGF gene and protein expression was determined using nonradioactive in situ hybridization and immunohistochemistry. On day 1, diseased animals developed focal mesangiolysis paralleled by a significant decrease in length density of glomerular capillaries that gradually returned to baseline levels thereafter, indicating capillary growth in response to initial injury. Glomerular VEGF mRNA expression increased on day 3 and returned back to baseline and beyond at day 14 when the glomerular recovery process was completed. Similarly, glomerular VEGF protein expression tended to be higher on day 3. The present study documents temporarily increased glomerular VEGF gene and protein expression during the healing of HSV GN, suggesting a potential role of VEGF in the repair of mesangiolytic glomerular damage.

Differential expression of genes in eutopic and ectopic endometrium from patients with ovarian endometriosis

To investigate whether genes that had been found to be differentially expressed in deep-infiltrating endometriosis and matched eutopic endometrium in our previous complementary DNA microarray study also are differentially expressed in ovarian endometriosis and matched eutopic endometrium. Prospective study. University hospital in France. Patients with ovarian endometriosis. During surgery, paired samples of tissue representing ovarian endometriosis and eutopic endometrium were obtained from 12 patients. Expression levels of messenger RNA for heat shock protein 90 alpha (HSP90A), chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2), prostaglandin E(2) receptor subtype EP3 (PGE(2)EP3), tyrosine kinase receptor B (TrKB), and 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2; epithelial cells) and of platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta 1 (PKCbeta1), Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase (MKK7), Sprouty2, mu-opioid receptor (MOR), and 5HTT (stromal cells) from ovarian endometriosis and matched eutopic endometrium were determined by using laser capture microdissection and real-time reverse-transcription polymerase chain reaction (RT-PCR) techniques. Expression of PDGFRA, PKCbeta1, JAK1, HSP90A, COUP-TF2, MOR, and 17betaHSD2 was significantly higher in ovarian endometriosis than in eutopic endometrium, whereas that of Sprouty2 and PGE(2)EP3 was significantly lower. There was no significant difference in mitochondrial RNA expression of MKK 7, TrKB, and 5HTT. Ovarian endometriosis might share several common molecules with deep-infiltrating endometriosis that act to sustain endometriotic lesions, whereas molecules involved in local endocrine control might be different between these two types of endometriosis.

Laser-capture microdissection

Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.

Myoepithelial carcinoma arising in an adenomyoepithelioma of the breast: Case report with immunohistochemical and mutational analysis

Adenomyoepithelioma (AME) of the breast is an uncommon tumor characterized by biphasic proliferation of both epithelial and myoepithelial cells. In rare instances, the epithelial, the myoepithelial or both components of an AME may become malignant. Described herein is the case of a 69-year-old woman who presented with myoepithelial carcinoma of the breast in an AME. Malignancy of myoepithelial component (MEC) was evidenced by the presence of cytological atypia, high mitotic rate, necrosis and local invasion. Immunohistochemical study demonstrated strong expression of P53 and phosphorylated extracellular signal-regulated kinase 1/2 in MEC. Laser capture microdissection technique and mutational analysis further revealed point mutation of the p53 gene (T-->G transversion at codon 270) in this population, but not in glandular epithelial cells or adjacent normal ductal epithelium. No mutations in exons 1 and 2 of the K-, H-, and N-ras genes were identified in any of the neoplastic component. To the authors' knowledge this is the first report of a mutation in the p53 gene in a malignant AME of the breast.

Mitochondrial Neurogastrointestinal Encephalomyopathy: Evidence of Mitochondrial DNA Depletion in the Small Intestine

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease clinically defined by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white-matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities. Loss-of-function mutations in thymidine phosphorylase gene induce pathologic accumulations of thymidine and deoxyuridine that in turn cause mitochondrial DNA (mtDNA) defects (depletion, multiple deletions, and point mutations). Our study is aimed to define the molecular basis of gastrointestinal dysmotility in a case of MNGIE. By using laser capture microdissection techniques, we correlated histologic features with mtDNA abnormalities in different tissue components of the gastrointestinal wall in a MNGIE patient and ten controls. The patient's small intestine showed marked atrophy and mitochondrial proliferation of the external layer of muscularis propria. Genetic analysis revealed selective depletion of mtDNA in the small intestine compared with esophagus, stomach, and colon, and microdissection analysis revealed that mtDNA depletion was confined to the external layer of muscularis propria. Multiple deletions were detected in the upper esophagus and skeletal muscle. Site-specific somatic point mutations were detected only at low abundance both in the muscle and nervous tissue of the gastrointestinal tract. Analysis of the gastrointestinal tract from 10 controls revealed a non-homogeneous distribution of mtDNA content; the small intestine had the lowest levels of mtDNA. Atrophy, mitochondrial proliferation, and mtDNA depletion in the external layer of muscularis propria of small intestine indicate that visceral myopathy is responsible for gastrointestinal dysmotility in this MNGIE patient.

In vivo association of CReMM/CHD9 with promoters in osteogenic cells

Molecular mechanisms that control cell differentiation involve with chromatin remodeling activities. We recently identified Chromatin Related Mesenchymal Modulator (CReMM), a CHD protein expressed by mesenchymal cells. In this study, we analyzed CReMM expression on RNA and protein levels during embryonic development in mouse skeletal tissues. CReMM appears transiently during mesenchymal cell differentiation, being detected first in osteoprogenitors and declining in mature cells. A novel aspect of the study elaborates on in vivo association of CReMM with promoters in cells obtained by laser capture micro-dissection (LCM) technique from periosteum and endochondreal ossification regions. Using chromatin immunoprecipitation (ChIP), we proved that CReMM binds to skeletal tissue-specific promoters: CBFA1, biglycan, osteocalcin (OC), collagen-II, and myosin in a differential manner. The results imply that CReMM selectively interacts with analyzed promoters activated in the tissue at the appropriate time of development. The identification of CReMM and its tissue distribution and function provides an attractive clue for the study of transcriptional regulation of osteogenic cells' maturation.

Methodology for Preservation of High Molecular-Weight RNA in Paraffin-Embedded Tissue

Laser-capture microdissection techniques have enhanced the ability to perform molecular studies of pure-cell populations. Although many technical factors affect the outcome of the procedure, none is more critical than the appropriate handling of the tissue. Because extraction of intact RNA from paraffin-embedded tissue is a difficult and inconsistent process, frozen sections with their attendant problems are used for this purpose. The major limitation of frozen section is its inferior morphologic quality compared with paraffin-embedded sections that may complicate accurate identification of cells during microdissection. We have developed a procedure that provides both high-quality histomorphology and RNA preservation in paraffin-embedded tissue. It is based on the use of a methanol-based fixative coupled with microwave-assisted rapid tissue processing. This technology in conjunction with a modified hematoxylin-eosin stain and a RNA extraction method allows isolation of high molecular-weight RNA from laser-capture microdissected, hematoxylin and eosin-stained paraffin sections. The high quality of the extracted RNA was confirmed by capillary electrophoresis and RT-PCR. The combination of a methanol-based fixative, rapid microwave tissue processing, and a modified hematoxylin and eosin stain produces paraffin sections that yield high molecular-weight RNA upon microdissection. This methodology opens the door for a wide range of gene expression analyses using paraffin-embedded tissue.

Combined small‐cell carcinoma of the stomach: p53 and K‐ras gene mutational analysis supports a monoclonal origin of three histological components

Primary small-cell carcinoma (SmCC) is extremely rare in stomach. We reported an autopsy case of combined gastric SmCC with p53 and K-ras mutational analysis. Histologically, the tumour was composed of well-differentiated adenocarcinoma surrounding the central dominant SmCC component with scattered nests of squamous cell carcinoma. Immunohistochemically, all the neoplastic components revealed strong expression for p53 protein. Based on these findings, we hypothesized that these histologically different components originated from a same progenitor cell that possessed p53 mutation. Using Laser-capture microdissection technique and mutational analysis, we identified the same point mutations of p53 gene (A-->G transversion in codon 239) and K-ras gene (G-->A transversion in codon 13) in all the neoplastic components, but not in the adjacent normal gastric epithelium. Our results strongly support a hypothesis that the combined SmCC of stomach in this case was of monoclonal origin.

Effect of mechanical stretch on HIF-1α and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study

Under physiological nonhypoxic conditions, angiogenesis can be driven by mechanical forces. However, because of the limitations of the specific gene expression analysis of microvessels from in vivo experiments, the mechanisms regulating the coordinated expression of angiogenic factors implicated in the process remain intangible. In this study, the technique of laser capture microdissection (LCM) was adapted for the study of angiogenesis in skeletal muscles. With a combination of LCM and real-time quantitative PCR it was demonstrated that capillary endothelial cells produce matrix metalloproteinase (MMP)-2 and that mechanical stretch of capillaries within muscle tissue markedly increases MMP-2 mRNA (2.5-fold increase vs. control; P < 0.05). In addition, we showed that transcription factor hypoxia-inducible factor (HIF)-1alpha expression was 13.5-fold higher in capillaries subjected to stretch compared with controls (P < 0.05). These findings demonstrate the feasibility of this approach to study angiogenic gene regulation and provide novel evidence of HIF-1alpha induction in stretched capillary endothelial cells.

Osteoclast-like cells express receptor activity modifying protein 2: application of laser capture microdissection

Receptor activity modifying proteins (RAMPs) act as receptor modulators that determine the ligand specificity of receptors for the calcitonin (CT) family. The purpose of this study was to analyze the expression of RAMPs in osteoclast-like cells using the laser capture microdissection (LCM) technique. Mouse bone marrow and spleen cells were co-cultured on a film designed for LCM. After 10 days, 250 osteoclast-like cells were captured using the LCM system. Total RNA from these cells was used to synthesize cDNA and RT-PCR analysis was performed. Osteoclast-like cells expressed CT receptor (CTR), CT receptor-like receptor (CRLR) and RAMP2, but did not express RAMP1 or RAMP3. These results indicated (1) that a pure population of osteoclast-like cells can be prepared by LCM and gene expression of this population can be analyzed by RT-PCR and (2) that RT-PCR shows that osteoclast-like cells express RAMP2, CTR and CRLR, suggesting the potential for adrenomedullin binding to osteoclast-like cells. This is the first report that osteoclast-like cells express RAMP2.

Mutations of mitochondrial 12S rRNA in gastric carcinoma and their significance

To detect the variations of mitochondrial 12S rRNA in patients with gastric carcinoma, and to study their significance and the relationship between these variations and the genesis of gastric carcinoma. PCR amplified mitochondrial 12S rRNA of 44 samples including 22 from gastric carcinoma tissues and 22 from adjacent normal tissues, was detected by direct DNA sequencing. Then laser capture microdissection technique (LCM) was used to separate the cancerous cells and dysplasia cells with specific mutations. Denaturing high performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw biosoft was used to analyze the RNA secondary structure of mutant-type 12S rRNA. Compared with Mitomap database, some new variations were found, among which np652 G insertion and np716 T-G transversion were found only in cancerous tissues. There was a statistic difference in the frequency of 12S rRNA variation between intestinal type (12/17, 70.59%) and diffusive type (5/17, 29.41%) of gastric carcinoma (P<0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutations. The frequency of 12S rRNA variation in cancerous cells was higher than that in dysplasia cells (P<0.01). 12S rRNA np652 G insertion showed obviously negative effects on the stability of 12S rRNA secondary structure, while others such as T-G transversion did not. The mutations of mitochondrial 12S rRNA may be associated with the occurrence of intestinal-type gastric carcinoma. Most variations exist both in gastric carcinomas and in normal tissues, and they might not be the characteristics of tumors. However, np652 G insertion and np716 T-G transversion may possess some molecular significance in gastric carcinogenesis. During the process from normality to dysplasia, then to carcinoma, 12S rRNA tends to convert from homoplasmy (wild type) to heteroplasmy, then to homoplasmy (mutant type, np717 T-G).

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.