Unlocking the archive - gene expression in paraffin-embedded tissue

Lewis, F A; Maughan, Nicola; Smith, Victoria; Hillan, Kenneth J.; Quirke, Philip

doi:10.1002/1096-9896(200109)195:1<66::aid-path921>3.0.co;2-f

Cited by 278 publications

(190 citation statements)

References 22 publications

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“…The most successful RNA extraction method to date was utilised in the present study (Lewis et al, 2001), but the yields were low and the RNA was without doubt degraded. Despite the compromised nature of the RNA obtained upon extraction from paraffin-embedded sections, highquality data has been previously obtained upon hybridisation to microarrays, with quantitative detection of up to 80% of genes when compared to fresh-frozen samples (Lewis et al, 2001). However, this is quite controversial and the use of RNA recovered from archival tissue samples for array analysis requires extensive validation.…”

Section: Discussionmentioning

confidence: 82%

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

et al. 2003

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p53 mutations and loss of heterozygosity have been commonly associated with oesophageal adenocarcinoma. In this investigation, the p53 status of a Welsh population of Barrett's-associated oesophageal adenocarcinomas were fully characterised at the gene sequence, chromosomal, mRNA and protein levels. In total, 31 tumours were examined for p53 gene sequence mutations using RFLP with sequencing, allelic loss of the gene was characterised by FISH, mRNA expression by p53 pathway signalling arrays and protein levels by p53 immunohistochemistry. In all, 9.6% of adenocarcinomas harboured p53 mutations, 24% displayed p53 allelic loss and 83% exhibited p53 protein accumulation. Point mutations and deletions of the gene did not coexist within the same samples. All samples containing p53 mutations also displayed positive immunostaining; however; in the majority of cases, p53 protein accumulation developed in the absence of mutations. The gene expression analysis demonstrated no differences in p53 and mdm-2 transcription levels between the p53 immunonegative and immunopositive samples, indicating other mechanisms underlie the proteins' overexpression. In conclusion, p53 mutations and deletions do not appear to be frequent events in oesophageal adenocarcinomas; however, abnormal accumulation of the protein is present in a vast majority of cases. P53 gene mutations are not the primary cause of protein overexpression -an alternative mechanism is responsible for the positive p53 immunohistochemistry detected.

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Section: Discussionmentioning

confidence: 82%

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

et al. 2003

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“…New molecular methods are currently being used for diagnostic purposes to detect eg, malignancy and infectious diseases. [11][12][13] Furthermore, Rish and co-workers 14 used a PCR method on FFPE tissue from mice experi-mentally infected with the H37Rv strain of M. tuberculosis and were able to detect as few as nine bacteria in a 5-m section of tissue. However, to obtain optimal DNA from such samples, several purification and preparation steps were needed.…”

mentioning

confidence: 99%

Detection of Mycobacterium tuberculosis Complex in Formalin-Fixed, Paraffin-Embedded Tissue Specimens with Necrotizing Granulomatous Inflammation by Strand Displacement Amplification

Johansen

Thomsen

Forsgren

et al. 2004

The Journal of Molecular Diagnostics

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“…The rationale for utilizing random hexamers comes from observations showing loss of the poly-T site due to increased susceptibility to degradation of RNA of the poly-A tail in extracted formalin-fixed and paraffin-embedded tissue. 15 Failure of reverse transcription and diminished successful recovery of RNA is regarded as secondary to loss of the priming site for poly-T. Random hexamers appear to facilitate successful RNA recovery both qualitatively and quantitatively. Adequate RNA extraction from formalin-fixed and paraffin-embedded tissue that facilitates successful subsequent gene expression analysis (including DNA microarray studies) is critical in our successful analysis of the DNA expression profiles of melanocytic lesions from formalin-fixed and paraffinembedded tissue.…”

Section: Discussionmentioning

confidence: 99%

“…14,15 Given that RNA quantitative studies have been successfully undertaken using formalin-fixed and paraffin-embedded tissue, their use for quantitative analysis of the DNA microarray system is of great interest, as tissue archives can provide a vast resource of desirable samples.…”

mentioning

confidence: 99%

Molecular classification of melanomas and nevi using gene expression microarray signatures and formalin-fixed and paraffin-embedded tissue

et al. 2009

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Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology. To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes. In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the feasibility of using molecular classification as a supplement to standard histology. Our successful use of a standard formalin-fixed and paraffin-embedded tissue further supports the practicability of combining molecular diagnostic testing with histopathology in evaluation of difficult melanocytic lesions.

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Unlocking the archive - gene expression in paraffin-embedded tissue

Cited by 278 publications

References 22 publications

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

Detection of Mycobacterium tuberculosis Complex in Formalin-Fixed, Paraffin-Embedded Tissue Specimens with Necrotizing Granulomatous Inflammation by Strand Displacement Amplification

Molecular classification of melanomas and nevi using gene expression microarray signatures and formalin-fixed and paraffin-embedded tissue

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