Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

Erickson, Heidi S.; Albert, Paul S.; Gillespie, John W.; Rodriguez‐Canales, Jaime; Linehan, W. Marston; Pinto, Peter A.; Chuaqui, Rodrigo F.; Emmert‐Buck, Michael R.

doi:10.1038/nprot.2009.61

Cited by 83 publications

(106 citation statements)

References 78 publications

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“…Microdissection-based quantitative mRNA analysis can contribute to the precise detection of MUC4 expression in tissue specimens [19][20][21]. Tissue microdissection techniques avoid the problem of cellular heterogeneity in favor of the molecular analysis of interesting cells without contamination by neighboring cells.…”

Section: Introductionmentioning

confidence: 99%

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma

et al. 2010

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The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.

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Section: Introductionmentioning

confidence: 99%

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma

et al. 2010

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“…Specifically, histopathological evaluation helps to identify the presence and amount of tumor cells, including type, patterns of invasion, and grade, as well as viability (vital vs. necrotic), and the presence and amount of other cell populations such as inflammatory cells and fibroblasts [35,39]. Furthermore, histopathological evaluation may also include the analysis of IHC staining patterns and levels of target protein expression.…”

Section: Box 1 Pathology Surgical Pathologists and Cytogeneticistsmentioning

confidence: 99%

“…Lastly, our own experience in developing and implementing laser capture microdissection (LCM) emphasizes the importance of pathological variables when working with tissue specimens for molecular cancer research [35,39,43]. LCM has become an important tool for analyzing tissue samples by facilitating a microdissection-based isolation or enrichment of specific cell types, such as cancer cells from complex specimens.…”

Section: Box 1 Pathology Surgical Pathologists and Cytogeneticistsmentioning

confidence: 99%

Why is it crucial to reintegrate pathology into cancer research?

Rodriguez‐Canales¹,

Éberlé²,

Jaffe³

et al. 2011

BioEssays

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The integration of pathology with molecular biology is vital if we are to enhance the translational value of cancer research. Pathology represents a bridge between medicine and basic biology, it remains the gold standard for cancer diagnosis, and it plays an important role in discovery studies. In the past, pathology and cancer research were closely associated; however, the molecular biology revolution has shifted the focus of investigators toward the molecular alterations of tumors. The reductionist approach taken in molecular studies is producing great insight into the inner workings of neoplasia, but it can also minimize the importance of histopathology and of understanding the disease as a whole. In turn, pathologists can underestimate the role of molecular studies in developing new ancillary techniques for clinical diagnosis. A multidisciplinary approach that integrates pathology and molecular biology within a translational research system is needed. This process will require overcoming cultural barriers and can be achieved through education, a more effective incorporation of pathology into biological research, and conversely an integration of biological research into the pathology laboratory.

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“…The resultant 20 μL of cDNA solution was diluted to 100 μL with distilled water. For real-time RT-PCR analysis, the quantity of DNA polymerase was increased versus the standard protocol to rapidly amplify aRNAs with a small copy number (4). Reagents were prepared as follows: FastStart Universal SYBR Green Master (Roche Diagnostics, Basel, Switzerland) 10 μL; FastStart Taq DNA Polymerase (5 U/ μL, Roche) 0.4 μL; forward Primer (10 μM) 2 μL; Reverse Primer (10 μM) 2 μL; distilled water 3.6 μL, aRNA solution 2 μL, and were run in triplicate on a Thermal Cycler Dice (Takara Bio, Otsu, Japan).…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

Determination of differential gene expression profiles in superficial and deeper zones of mature rat articular cartilage using RNA sequencing of laser microdissected tissue specimens

et al. 2014

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Superficial zone (SFZ) cells, which are morphologically and functionally distinct from chondrocytes in deeper zones, play important roles in the maintenance of articular cartilage. Here, we established an easy and reliable method for performance of laser microdissection (LMD) on cryosections of mature rat articular cartilage using an adhesive membrane. We further examined gene expression profiles in the SFZ and the deeper zones of articular cartilage by performing RNA sequencing (RNA-seq). We validated sample collection methods, RNA amplification and the RNA-seq data using real-time RT-PCR. The combined data provide comprehensive information regarding genes specifically expressed in the SFZ or deeper zones, as well as a useful protocol for expression analysis of microsamples of hard tissues.Osteoarthritis (OA), a chronic degenerative joint disorder characterized by articular cartilage destruction, is a major public health issue, causing pain and disability of the elderly worldwide (5, 8). Although effective disease-modifying treatments for OA have not been developed, its etiopathogenesis has been addressed in recent clinical studies. Cartilage degeneration is first observed at the articular surface in the form of fibrillation (24). Once the surface is disrupted, deeper cartilage layers are subsequently degraded (24). Articular cartilage (AC) is composed of three layers: the superficial (SFZ), middle and deep zones. The SFZ, the outermost surface layer adjacent to the joint cavity, is histologically distinct from the deeper zones. In the SFZ, collagen fibers align parallel to the articular surface, in contrast to their vertical alignment in the deeper layers (2, 17). SFZ cells, which also display parallel alignment, are smaller than chondrocytes in the deeper layers and exhibit characteristic flat morphology (10). SFZ cells produce lubricin, encoded by Proteoglycan4 (Prg4), for surface lubrication. An homozygous mutant of the human PRG4 gene causes the autosomal recessive camptodactyly-arthropathy-coxa vara-pericarditis syndrome that shows congenital or early-onset camptodactyly and childhood-onset noninflammatory arthropathy (1, 16). Mice lacking Prg4 (Prg4−/−) exhibit early onset of osteoarthritis (25). In addition, chondrocytes obtained from SFZ display higher proliferative activity than those from deeper AC zones, implying that SFZ might be the main cell source of cartilage regeneration (32). Despite the potential roles of SFZ in the etiopathogenesis and pathophysiology of OA, apart from the involvement of Wnt/ β-catenin signaling, TGF-β/BMP signaling and high mobility group box 2 (HMGB2), molecular mechanisms regulating the differentiation and maintenance of SFZ are still unknown (12, 22, 28). This lack of data is mainly because comprehensive in vivo gene expression analysis of the SFZ is difficult due to its thinness, which makes it difficult to obtain SFZ-spe- layer of liquid nitrogen until microdissection. To prevent RNA degradation, frozen specimens (prior to thawing) were immediately soaked in 8...

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Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

Cited by 83 publications

References 78 publications

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma

Why is it crucial to reintegrate pathology into cancer research?

Determination of differential gene expression profiles in superficial and deeper zones of mature rat articular cartilage using RNA sequencing of laser microdissected tissue specimens

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