Molecular biological analysis of paraffin-embedded tissues

Mies, Carolyn

doi:10.1016/0046-8177(94)90218-6

Cited by 87 publications

(39 citation statements)

References 35 publications

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“…imens (Mies, 1994). However, RT-PCR amplification of RNA from this material is a complex procedure that may be adversely influenced by several factors, mainly by nucleic acid breakdown during tissue processing and storage (Aurer et al, 1993).…”

Section: Soguero Et Almentioning

confidence: 99%

Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

Soguero

Campo

Ribalta

et al. 2000

Laboratory Investigation

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SUMMARY:Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5Јnon-coding region (5ЈNCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5ЈNCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5ЈNCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5ЈNCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5ЈNCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV. (Lab Invest 2000, 80:851-856).

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Section: Soguero Et Almentioning

confidence: 99%

Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

Soguero

Campo

Ribalta

et al. 2000

Laboratory Investigation

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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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“…Breakpoints in the linear series of bases in DNA prevent oligonucleotide-directed enzymatic amplification of DNA despite primer annealing in the context of an appropriately optimized PCR reagent mixture, thus leading to the occurrence of false negative amplification results (Mies 1994). However, partially degraded low molecular weight DNA from formalin-fixed paraffin-embedded tissue can serve adequately in PCR, provided that yields of DNA from extraction techniques are sufficient and that primer strategies target smaller regions of DNA for amplification (Honma et al 1993, Mies 1994. Initial attempts at PCR in this study were unsuccessful using primers designed to amplify an 858-base pair fragment of the myxosporean SSU rDNA in either single-round or nested protocols (data not shown).…”

Section: Polymerase Chain Reaction and Hybridization Experimentsmentioning

confidence: 99%

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

Frasca¹,

Linfert²,

Tsongalis³

et al. 1999

Dis. Aquat. Org.

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'~e p a r t m e n t of Pathobiology. U-89, University of Connecticut. Storrs. Connecticut 06269-3089. USA '~e p a r t m e n t of Pathology and Laboratory Medicine, Hartford Hospital. Hartford, Connecticut 06102. USA 3~e p a r t m e n t of Medicine and Epidemiology, School of Veterinary Medicine, One Shields Avenue, University of California. Davis, California 95616, USA 4Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555. USA ABSTRACT: During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozolc myxosporean, possibly a A4yxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybrihzation, &deoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide pnmers were created from sequences of the SSU rRNA gene of A4. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light rnicroscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doon, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothe...

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Molecular biological analysis of paraffin-embedded tissues

Cited by 87 publications

References 35 publications

Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

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

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

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