scholarly journals Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR

2020 ◽  
Vol 21 (14) ◽  
pp. 5119
Author(s):  
Takeshi Shimizu ◽  
Toshitsugu Fujita ◽  
Sakie Fukushi ◽  
Yuri Horino ◽  
Hodaka Fujii
Keyword(s):  
Methylation Status ◽  
Pcr Amplification ◽  
Cpg Methylation ◽  
Target Sequence ◽  
Tissue Specimen ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Blood Specimens ◽  
Formalin Fixed

Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.

scholarly journals Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tim A. D. Smith ◽  
Omneya A. AbdelKarem ◽  
Joely J. Irlam-Jones ◽  
Brian Lane ◽  
Helen Valentine ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Candidate Genes ◽  
Pcr Amplification ◽  
The Cancer Genome Atlas ◽  
Analysis Tool ◽  
Endogenous Control ◽  
Formalin Fixed Paraffin ◽  
Stability Ranking ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

scholarly journals Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

2006 ◽  
Vol 54 (7) ◽  
pp. 773-780 ◽  
Author(s):  
Kiyohiro Hamatani ◽  
Hidetaka Eguchi ◽  
Keiko Takahashi ◽  
Kazuaki Koyama ◽  
Mayumi Mukai ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rt Pcr ◽  
Molecular Analyses ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

Immunohistochemical Detection of Canine Adenovirus in Paraffin Sections of Liver

1986 ◽  
Vol 23 (4) ◽  
pp. 478-484 ◽  
Author(s):  
P. M. Rakich ◽  
K. W. Prasse ◽  
P. D. Lukert ◽  
L. M. Cornelius
Keyword(s):  
Immunohistochemical Detection ◽  
Hepatic Inflammation ◽  
Paraffin Sections ◽  
Inflammatory Lesions ◽  
Long Term Stability ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

An avidin-biotin immunoperoxidase procedure was optimized for detection of canine adenoviral antigens in formalin-fixed, paraffin-embedded liver. Long-term stability of viral antigen was shown by successful demonstration of virus in liver tissue preserved up to six years from dogs with infectious canine hepatitis. This immunohistochemical stain was applied to sections from livers with a wide range of inflammatory lesions. Examination of sections from 53 dogs yielded five livers with small amounts of adenovirus. An additional virus-positive liver was identified from a dog with no hepatic inflammation. Although a cause and effect relationship remains to be determined, these findings suggest a possible connection between canine adenovirus and spontaneous chronic hepatitis.

scholarly journals NeuN: a useful neuronal marker for diagnostic histopathology.

1996 ◽  
Vol 44 (10) ◽  
pp. 1167-1171 ◽  
Author(s):  
H K Wolf ◽  
R Buslei ◽  
R Schmidt-Kastner ◽  
P K Schmidt-Kastner ◽  
T Pietsch ◽  
...  
Keyword(s):  
Neuronal Cell ◽  
Cell Types ◽  
Specific Protein ◽  
Antigen Retrieval ◽  
Neuronal Marker ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Diagnostic Histopathology

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

scholarly journals BCL-1 Gene Rearrangements in Iranian Non-Hodgkin Lymphoma Patients

2015 ◽  
Vol 8 (8) ◽  
pp. 108
Author(s):  
Manoush Tohidirad ◽  
Mehrdad Asghari Estiar ◽  
Azim Rezamand ◽  
Saeid Ghorbian ◽  
Sasan Andalib ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Pcr Amplification ◽  
B Cell Lymphoma ◽  
Gene Rearrangements ◽  
Non Hodgkin Lymphoma ◽  
Formalin Fixed Paraffin ◽  
Mantle Cell ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

<p>In the present study, our aim was to assess the incidence of BCL-1 gene rearrangements in formalin-fixed paraffin embedded (FFPE) tissue in patients with non-Hodgkin lymphomas (NHL). The BIOMED-2 protocol was applied to assess the BCL-1 gene rearrangements in NHL patients. PCR amplification was carried out on FFPE in 100 patients with B-cell lymphoma including 89 cases with diffused large B-cell lymphoma (DLBCL) (15 cases under 18 years old) and 11 cases with mantle cell lymphoma (MCL). Out of the 100 patients, 19 cases (19%) were identified to have concurrent translocation involving BCL-1. The significant association was seen between BCL-1 gene rearrangements and the lymphomas in patients older than 55 years (P&lt;0.05). Out of 100 cases, 80 cases were positive and 20 cases were negative regarding CD20. No significant association was found between DLBCL lymphoma in patients under 18 years old and BCL-1 gene rearrangements (P&gt;0.05). In addition, the positive and negative expressions of LCA/CD45 marker were 76% (76/100) and 26% (26/100), respectively. Our findings revealed that BCL-1 gene rearrangement assays using BIOMED-2 protocol can be considered as a valuable approach in detection of the lymphomas.</p>

scholarly journals p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

1992 ◽  
Vol 40 (7) ◽  
pp. 1047-1051 ◽  
Author(s):  
B J Kerns ◽  
P A Jordan ◽  
M B Moore ◽  
P A Humphrey ◽  
A Berchuck ◽  
...  
Keyword(s):  
Breast Cancers ◽  
Fresh Tissue ◽  
P53 Overexpression ◽  
P53 Expression ◽  
Ovarian Cancers ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

scholarly journals KIT Somatic Mutations and Immunohistochemical Expression in Canine Oral Melanoma

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2370
Author(s):  
Ginevra Brocca ◽  
Beatrice Poncina ◽  
Alessandro Sammarco ◽  
Laura Cavicchioli ◽  
Massimo Castagnaro
Keyword(s):  
Pcr Amplification ◽  
Mean Value ◽  
Single Nucleotide ◽  
Oral Melanoma ◽  
Pathway Activation ◽  
Formalin Fixed Paraffin ◽  
Frequent Event ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 11 ◽  
Formalin Fixed

Canine oral melanoma (COM) is an aggressive neoplasm with a low response to therapies, sharing similarities with human mucosal melanomas. In the latter, significant alterations of the proto-oncogene KIT have been shown, while in COMs only its exon 11 has been adequately investigated. In this study, 14 formalin-fixed, paraffin-embedded COMs were selected considering the following inclusion criteria: unequivocal diagnosis, presence of healthy tissue, and a known amplification status of the gene KIT (seven samples affected and seven non-affected by amplification). The DNA was extracted and KIT target exons 13, 17, and 18 were amplified by PCR and sequenced. Immunohistochemistry (IHC) for KIT and Ki67 was performed, and a quantitative index was calculated for each protein. PCR amplification and sequencing was successful in 97.62% of cases, and no single nucleotide polymorphism (SNP) was detected in any of the exons examined, similarly to exon 11 in other studies. The immunolabeling of KIT was positive in 84.6% of the samples with a mean value of 3.1 cells in positive cases, yet there was no correlation with aberration status. Our findings confirm the hypothesis that SNPs are not a frequent event in KIT activation in COMs, with the pathway activation relying mainly on amplification.

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