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Microdissection, microchip arrays, and molecular analysis of tumor cells (primary and metastases).

Advances in biotechnology and bioinformatics are offering promise for new breakthroughs in gene discovery and elucidation of gene function. At present, many candidate genes related to cancer pathogenesis have been identified in several types of human cancer, yet frequently their function remains elusive. This is particularly true as it relates to the progression of human cancer. This landscape could change dramatically, however, as technological innovations and improvements continue to revolutionize these fields. High-throughput molecular approaches are emerging, which may become accurate, automated, and cost-effective. For example, DNA arrays on microchips are under development with numerous applications, including the ability to screen genes rapidly for mutations and to study patterns of gene expression on a large scale. Automated systems for microdissection and sequencing are also in their implementation stages. Commensurate with their integration and evolution, these information and technological tools have the potential to offer a more comprehensive understanding of multiple genetic and cellular alterations occurring during cancer initiation, development, and progression. Ultimately, this fundamental knowledge can provide strategies for intervention, prevention, and early diagnosis. This is a US government work. There are no restrictions on its use.

Base Sequence↗

DNA alterations during multi-step development of human hepatocellular carcinomas revealed by laser capture microdissection.

In order to clarify early molecular events involved in liver carcinogenesis, we analyzed 53 liver-cirrhosis nodules (LCNs) from five patients and 13 micro-hepatocellular carcinoma (HCC) nodules from one patient and looked for alterations of microsatellites in genomic DNA after carefully preparing the tissue samples by laser-capture microdissection (LCM). Allelotyping was done with 20 markers corresponding to anonymous microsatellites and 13 corresponding to tumor suppressor genes (TSGs) that had shown significant alterations in HCCs. We detected both loss of heterozygosity (LOH) and microsatellite shifts (MS). Overall, 24 of 53 (47%) of LCNs showed LOH with any of the informative markers used in the study, reflecting that proportion of LCNs with clonal growth. The fractional allelic loss (FAL) index, an indication of total genomic complexity, was not significantly different between LCN and micro-HCC nodules, but their profiles of alteration were different. These profiles were classified into three groups: (1) LCN profile-allelic loss at chromosomal arms 1q and 14q, TBP and BRCA1; (2) HCC profile-LOH at 4q, 6q, 7q, 17p, NF1, IGFIIr and p53 in micro-HCC nodules; these changes in early lesions were identical to those seen in mature HCCs; (3) Common profile-LOH at NF1 and 6q, including IGFIIr, common to both LCN and HCC. No LCN showed LOH at p53 and Rb, loci that are generally altered in HCCs. However, 12 intra-tumoral nodules examined had lost p53 in all informative cases, although the loss of Rb was a late event. These results suggest that early genomic profiles confined to LCNs, and additional profiles that can be observed when liver tissue undergoes malignant transformation, support a model of multi-step development of HCC.

Journal Article↗

Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis.

Gene expression profiling using high density oligonucleotide arrays is a powerful method to generate an unbiased survey of a cell's transcriptional landscape. Increasingly complex biological questions require that this approach be applicable to the small numbers of cells that are obtained from sources such as laser capture microdissection (LCM) of solid tissues. In this report, we demonstrate that two rounds of transcript amplification can generate accurate and reproducible gene expression profiles using high density oligonucleotide microarrays, starting with as little as 10 ng of total RNA. Biased amplification of the 3' end of transcripts does not have a major impact on the overall transcript profile due to the 3' bias of probe sets incorporated in the array design. Furthermore, greater than 95% of all genes detected demonstrate less than a twofold difference in expression when independent tissue dissections of identical cell populations are compared. The accuracy and technical reproducibility of the method suggests that expression profiling using transcript amplification and high density oligonucleotide microarrays can be used on a routine basis.

Breast Neoplasms↗

Multiplex genotype analysis of invasive carcinoma and accompanying proliferative lesions microdissected from breast tissue.

To understand the genetic basis of breast cancer in a comprehensive way, purported precursor lesions need to be analyzed at a large number of genetic marker loci and compared with each other and with the invasive components. However, the microscopic size of most of these lesions and the very small amount of material that can be obtained through microdissection limit the number of loci that can be included in the analysis. To address this issue, a multiplex genotyping approach has been developed. With this approach, polymorphic sequences at 28 marker loci were amplified simultaneously from the micro-dissected components in 5-microm paraffin-embedded breast tissue sections. The genotypes of the lesions were determined after resolving the amplified allelic products by denaturing gradient gel electrophoresis. Because the material isolated from each lesion in a single 5-microm section was sufficient for several 28-locus assays and several successive tissue sections with the same set of lesions may be prepared, it is possible to determine the genotype of each lesion at hundreds of genetic marker loci that may well cover the human genome. Analyzing a sufficient number of cases may yield information that could be used to understand the genetic basis of breast cancer development in a comprehensive way.

Alleles↗

Quantitative amplification of genomic DNA from histological tissue sections after staining with nuclear dyes and laser capture microdissection.

Laser capture microdissection (LCM) allows the selective sampling of tissue from histological sections. A prerequisite for this technique is the availability of histological dyes that do not interfere with downstream analysis of the sampled genetic material. We have examined the effect of four histological nuclear dyes (methyl green, hematoxylin, toluidine blue O, azure B) on TaqMan polymerase chain reaction amplification of beta-actin genomic DNA prepared from fixed and frozen tissue. Tissue sampled from the histological sections by manual dissection was compared with tissue sampled by LCM. As previously reported, when manually dissected tissue sections were analyzed, polymerase chain reaction amplification of DNA after hematoxylin staining was inferior to that after staining with the other dyes. In contrast, when tissue sampled by LCM was examined, DNA recovery after hematoxylin staining was equivalent to the recovery after methyl green staining. We conclude that DNA recovery from LCM-sampled tissue is independent of the histological stain chosen to highlight nuclear detail.

Cell Nucleus↗

Proteome analysis of microdissected tumor tissue using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-tandem MS.

This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high-confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 microg of total protein, an amount corresponding to 20,000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of CIEF fractions without an accompanying increase in sample consumption.

Brain Neoplasms↗

Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels.

We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 microg renal cell carcinoma protein and 3.8 microg normal kidney protein respectively from one patient. The study involved separate radio-iodination of each sample with both (125)I and (131)I, dual inverse replicate sample loading to high resolution 54 cm "daisy chain" serial immobilized pH gradient isoelectric focusing (IPG-IEF) 2D-PAGE gels, co-electrophoretic separation of cross-labeled proteins from different samples, and precision multiplex differential radioactive imaging to obtain signals specific for each sample coelectrophoresed within single gels but labeled with different isotopes of iodine, providing extremely precise intra-gel estimates of the abundance ratio for protein spots from both samples. Twelve multiplexed analytical radioactive SDS-gels from 4 serial IPG-IEF gels provided 24 individual radioactive images for a comprehensive analytical protein multiplex quantification study. A further 12 SDS gels containing (125)I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed <40% of the (125)I-labeled sample, and <20% of the (131)I-labeled sample from the respective original 3.8 microg samples. Twenty-nine proteins were identified by mass spectrometry with PMF scores >70 that were >2-fold differentially abundant between the samples and t-test probabilities <0.05. We conclude that this combination of technologies provides excellent quality protein multiplex data for the differential abundance analysis of large numbers of proteins from extremely small samples, and is applicable to a broad range of clinical and related applications.

Carcinoma, Renal Cell↗

Microdissected bovine X chromosome segment delineates homoeologous chromosomal regions in sheep, goat and buffalo.

The terminal part of the long arm of the bovine X chromosome (bands Xq41-q43) was microdissected. The DNA thus obtained was PCR amplified, labelled and used as painting probe on cattle, sheep, goat and buffalo chromosomes. In cattle, as expected, distinct hybridization signals were observed on bands Xq41-q43. In sheep and goat, the painting signals were observed on the proximal part of the long arm of the X chromosome, adjacent to the centromere (Xp12-q12). In buffalo, however, the terminal part of the X chromosome involving bands q44-q47 was painted. The findings contribute towards developing a better understanding of the comparative organization of the X chromosome in the four bovidae species. Proposed models of evolutionary rearrangements within the X chromosome of the four species are examined in light of the results obtained.

Animals↗

XY sex reversal in the wood lemming is associated with deletion of Xp21-23 as revealed by chromosome microdissection and fluorescence in situ hybridization.

In the wood lemming (Myopus schisticolor), XY sex reversal occurs naturally because of the presence of an X chromosome variant designated X*. The two types of X chromosome, X and X*, can be distinguished by G-banding, and analyses have demonstrated complex rearrangements of the short arm of X*. Here, chromosomal microdissection, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and fluorescence in situ hybridization (FISH) techniques have been used to generate and map DNA probes for different parts of the X and X* chromosomes. The results showed that the region of Xp21-23 is deleted from the X* and some of the deleted DNA sequences are homologous to the mouse gamma-satellite. The deletion must be associated with the sex reversal in this species. FISH experiments with dissected probes of X and distal half of Xq provided evidence for presence of homologous sequences between large regions of the X and Y chromosomes, including euchromatic and heterochromatic parts of the sex chromosomes. The findings of this study will be of significance for further cloning of important candidate gene(s) responsible for the XY sex reversal.

Animals↗

Meiotic chromosomes as templates for microdissection.

As templates for chromosome microdissection, meiotic cells offer several advantages over mitotic cells. The pairing of homologous chromosomes at the metaphase plate of the first meiotic division allows the simultaneous isolation of two copies of the same chromosome, and the sex chromosomes are easy to identify in male meiotic cells. We report on a method for making fluorescence in-situ hybridization (FISH) probes from dissected meiotic chromosomes.

Animals↗

Isolation of DNA from the centromere of human chromosome 7 by microdissection.

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a microdissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3-4cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.

Amniotic Fluid↗

The use of laser microdissection for the preparation of chromosome-specific painting probes in farm animals.

Laser microbeam microdissection and laser pressure catapulting procedure were used for the construction of chromosome-specific painting probes, arm-specific probes and probes for chromosomal subfragments. We report on a method for generation of fluorescence in-situ hybridization probes from laser dissected chromosomes of farm animals. So far, using the described method, a set of chromosome-specific painting probes has been obtained for all porcine chromosomes, 17 chromosomes of cattle and selected equine chromosomes. It is concluded that the laser technology appears to be a useful and powerful tool for the construction of chromosome-specifi c painting probes. Its main advantage is the fast non-contact collection of chromosomes.

Animals↗

A probe generated by chromosome microdissection, useful for analyzing Y chromosome evolution in Old World monkeys.

We isolated a DNA probe, designated MMDYZ1, using a chromosome microdissection technique from the Y chromosome of the Rhesus monkey. The probe obtained from eight whole Y chromosomes shows higher specificity for the Y short arm of the Rhesus monkey, which consists totally of constitutive heterochromatin. Two microclones (MMY#3 and MMY#4) were constructed from the Y-specific primary PCR products. Sequence analysis of these two microclones revealed that both were essentially identical to each other and the sizes were 870 and 686 bp, respectively. From alignment analysis using the Genbank database of primates, the alphoid DNA has the highest affinity with the probe. However, the total composition of this probe has extremely high homology with the Y short arm of the Rhesus monkey, as demonstrated by fluorescence in-situ hybridization (FISH). Comparative FISH-mapping disclosed that this DNA-sequence cluster was located at extremely different sites on the Y chromosome in several species of the Old World monkey. Accordingly, this probe seems to be a high-quality tool, now established for the first time, for investigating Y chromosome evolution of the Old World monkey.

Animals↗

Caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells in mutant SOD1 transgenic mouse spinal cord: a study using laser microdissection and real-time RT-PCR.

Amyotrophic lateral sclerosis is characterized by selective motor neuron degeneration. An apoptotic pathway is thought to be involved. It is difficult, however, to analyze the molecular pathogenic mechanism in single motor neurons because of complexity in the neural tissue, which consists of multiple lineages of cells neighboring motor neurons. We quantified the caspase-1 and -3 mRNA in single motor neurons and neighboring glial cells isolated from the spinal ventral horn of mutant SOD1 transgenic (Tg) mice and littermates. Motor neurons and neighboring glial cells were isolated from spinal sections by laser microdissection, and the mRNAs were quantified by RT-PCR. In the Tg mice, caspase-1 mRNA was first upregulated in motor neurons and second in glial cells. The caspase-3 mRNA was increased in motor neurons following the caspase-1 mRNA. These results indicated that caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells of the Tg mice, and that mRNAs in isolated cells can be accurately assessed using our procedures.

Animals↗

Calcitonin receptor gene and breast cancer: quantitative analysis with laser capture microdissection.

There is a growing body of evidence indicating that calcitonin (CT) and its receptor (CTR) is involved in cell growth, differentiation and tissue development. Using laser capture microdissection (LCM) and real-time reverse transcription polymerase chain reactions (RT-PCR), we have investigated CTR mRNA expression in 60 primary breast cancers, including 14 pairs of matched cancers and unaffected ductal epithelia from the same patients. Our results demonstrate that CTR mRNA was constantly expressed in normal ductal epithelium and in breast cancer. In the 14 cases where matched samples were available, a decrease in CTR mRNA expression was found in 9 breast cancers (64.3%), an increased CTR expression in 2 cases (14.3%) and no significant change in 3 cases (21.4%). In 60 cases of primary breast cancers, decreased CTR expression was found in 44 (73.3%), increased CTR expression was detected in 10 cases (16.7%) and no change was observed in 6 cases (10%). Decreased CTR expression was found more often in cases with lymph node metastasis (p = 0.0498) and lymphatic invasion (p = 0.0179). Also there was a decreased CTR expression in cases with an extensive intraductal component (p = 0.0543) and a high nuclear grade (p = 0.1934), although this was not statistically significant. Overall, we conclude that CTR mRNA was constantly expressed in unaffected ductal epithelium, whereas decreased CTR mRNA expression was frequently found in breast cancers, particularly in cases with lymph node metastasis and lymphatic invasion. These results suggest that CTR might be of great potential significance in breast cancer progression.

Adult↗

[Microdissection of chromosomes in veterinary medicine].

Microdissection of chromosomes and subsequent amplification of the DNA material by PCR allow the development of libraries, region-specific DNA probes or chromosome painting probes. Veterinary medicine will benefit from this method in instances where clarification of numercal or structural chromosome aberrations is needed.

Animals↗

Isolation of 24 novel cDNA fragments from microdissected human chromosome band.

The strategy of isolating the band-specific expression fragments from a probe pool generated by human chromosome microdissection was reported. A chromosome 14q24.3 band-specific single copy DNA pool was constructed based on this probe pool. Using total DNA of the pool as probe to hybridize the human marrow cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Among these primary clones, 32 secondary clones were obtained after second-round screening and designed as cFD14-1-32. Finally, 24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization. Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but can't hybridize to 17q11-12 DNA. Partial sequences of 13 fragments of them were sequenced and identified as novel cDNA sequences, and these sequences were proved to have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

Blotting, Northern↗

Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection.

A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification, characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method. Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp. This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.

Chromosome Mapping↗