DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.     

Detection of HER2/neu gene amplification in archival paraffin-embedded breast cancer tissues by fluorescence in situ hybridization

We evaluated HER2/neu gene amplification by fluorescence in situ hybridization (FISH) in archival paraffin-embedded breast cancer tissues. Tumors from 63 human invasive breast cancers were categorized into two groups depending on whether the paraffin-embedded tissue blocks had been stored for more or less than 12 months duration. These were subjected to routine and modified FISH protocols. As microwave oven formalin fixation of tissues was carried out in the majority of the older archived specimens, the effect of this fixation method was also analyzed. FISH signals were obtained in all 13 archival specimens stored for less than 12 months. However, in 50 specimens stored for more than 12 months duration, the procedure was successful in only 10 specimens (20%), for which the pretreatment procedure had to be individually optimized for each specimen. There was no significant difference in the detection of FISH signals between microwave oven and routinely fixed specimens.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.     

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.     

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/ VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22–79, PHM, prepro-VIP 111–122, VIP and prepro-VIP 156–170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single-or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.     

Detection of Helicobacter pylori and determination of clarithromycin susceptibility using formalin-fixed, paraffin-embedded gastric biopsy specimens by fluorescence in situ hybridization

BACKGROUND: Clarithromycin resistance and poor compliance to therapy are often responsible for Helicobacter pylori eradication therapy failure. AIM: To evaluate fluorescence in situ hybridization (FISH) as a nonculture method to simultaneously detect H. pylori and to identify clarithromycin resistance. METHODS: Fifty-four patients with dyspepsia (17 male, 37 female subjects; mean age, 46.5; range, 21-78 years) were studied. Two antrum and corpus biopsies were taken from each patient. Positive rapid urease test (RUT) and histopathologic examinations defined H. pylori positivity. A total of 108 formalin-fixed paraffin-embedded gastric mucosal biopsies were examined retrospectively by the FISH (seaFAST H. pylori Combi-Kit) method. RESULTS: Forty-five patients (83.3%) were H. pylori positive and 43 (95.5%) were also positive by FISH. There were two false-positive FISH results. Fourteen patients (31.1%) had clarithromycin-susceptible strains, 4 (8.9%) resistant strains, and 27 (60%) both susceptible and resistant strains. CONCLUSION: FISH results correlated well with H. pylori infection and were able to identify clarithromycin-susceptible and -resistant strains. This technique will be helpful in determining the bacterial density and the success of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

PCR detection of Actinobacillus pleuropneumoniaeapxIV gene in formalin-fixed,paraffin-embedded lung tissues and comparison with in situ hybridization

AIMS: Formalin-fixed, paraffin-embedded lung tissues from pigs experimentally infected with 12 Actinobacillus pleuropneumoniae serotypes were used to develop nested PCR for the detection of apxIV gene. METHODS AND RESULTS: The PCR results from formalin-fixed, paraffin-embedded tissues were compared with in situ hybridization. The apxIV gene was detected in formalin-fixed, paraffin-embedded lung tissues from all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes by nested PCR. In situ hybridization produced a distinct positive signal in all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes. Agreement rates between nested PCR and in situ hybridization were 100% for the detection of apxIV gene in formalin-fixed paraffin-embedded lung tissues. Acceptable PCR signals were detected from lung tissues fixed for periods up to 180 days. CONCLUSIONS: The apxIV gene is species-specific rather than serotype-specific and is therefore an important diagnostic marker. The nested PCR assay would be a useful method for the detection of apxIV gene to diagnose A. pleuropneumoniae infection when formalin-fixed tissues are submitted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirmed the possibility of using formalin-fixed, paraffin-embedded tissues for the diagnosis of A. pleuropneumoniae infection in pigs.     

Dihydrotestosterone (DHT) regulation of insulin-like growth factor II mRNA in neonatal rats

Insulin-like growth factors (IGFs) are well known as peptide mitogens and important growth factors in fetal as well as in early postnatal development. In particular, IGF II is strongly expressed during fetal life and in neonatal animals. Very little is known about the regulation of IGF II gene expression. In order to study in detail the regulation of IGF II mRNA levels in the liver by the potent nonaromatizable androgen dihydrotestosterone (DHT), we have used quantitative in situ hybridization to detect the mRNA encoding for this growth factor. Pups were separated into 4 groups and injected twice a day immediately after birth with 3 different doses of DHT: 0.1 mg DHT/day, 0.25 mg DHT/day, 0.5 mg DHT/day for 4 and 7 days, and the control groups were injected with the vehicle alone. Animals were perfused with 4% paraformaldehyde and sections from the liver, heart, kidneys and brain were cut with a cryostat. A [³⁵S]-labeled cDNA probe was used to detect IGF II mRNA levels. After hybridization, sections were autoradiographed with X-ray films and then coated with liquid photographic emulsion. Densitometric measurement revealed that, at 4 days of age, IGF II mRNA levels were lower in DHT-treated rats than in control animals. No statistically significant differences in IGF II mRNA levels were observed among the three groups treated with the different doses of DHT, thus revealing that even the lowest dose of DHT (0.1 mg/day) used was sufficient to inhibit IGF II gene expression in neonatal rats. Moreover, at 7 days of age, DHT-treated rats showed the same levels of IGF II mRNA as those observed in rats treated with DHT for 4 days. These results suggest that DHT may play an important role in the regulation of IGF II gene expression in the rat liver during the neonatal period.     

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed,paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.     

Identification of differentially expressed genes in omental adipose tissues of obese patients by suppression subtractive hybridization

To identify differentially expressed genes between obese individuals and normal control, we have undertaken suppression subtractive hybridization (SSH). Omental adipose tissues were obtained via abdominal surgery for appendicitis in both 13 obese subjects [BMI (body mass index) >30 kg/m2] and 13 normal subjects (BMI >18 and <25 kg/m2). Following SSH, about one thousand clones were sequenced and found to derive from 426 different genes. These predominately expressed genes included genes involved in lipid metabolism, cytokines, signal transduction, GLUT4 translocation, cell cycle and growth, cytoskeleton, and others. Although more detailed analyses are necessary, it is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of obesity.