scholarly journals Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression

2010 ◽  
Vol 56 (12) ◽  
pp. 1845-1853 ◽  
Author(s):  
Guido Hennig ◽  
Mathias Gehrmann ◽  
Udo Stropp ◽  
Hiltrud Brauch ◽  
Peter Fritz ◽  
...  
Keyword(s):  
Extraction Method ◽  
Nucleotide Polymorphisms ◽  
Automated Extraction ◽  
Single Nucleotide ◽  
Normal Tissues ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna And Dna ◽  
Formalin Fixed

BACKGROUND There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. METHODS We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1–25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. RESULTS In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%–100% of tumor tissues and 21%–100% of normal tissues. The 7 SNPs were successfully genotyped in 91%–97% of tumor and 94%–97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%–99.5%. CONCLUSIONS This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

scholarly journals Higher EU-TIRADS-Score Correlated with BRAF V600E Positivity in the Early Stage of Papillary Thyroid Carcinoma

2021 ◽  
Vol 10 (11) ◽  
pp. 2304
Author(s):  
Karolina Skubisz ◽  
Joanna Januszkiewicz-Caulier ◽  
Patrycja Cybula ◽  
Elwira Bakuła-Zalewska ◽  
Krzysztof Goryca ◽  
...  
Keyword(s):  
Early Stage ◽  
Braf V600e ◽  
P Value ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ion Proton ◽  
Indolent Disease ◽  
Next Generation Sequencing Ngs ◽  
Formalin Fixed

The data demonstrating a correlation between sonographic markers of malignancy of thyroid cancer (TC) and its genetic status are scarce. This study aimed to assess whether the addition of genetic analysis at the preoperative step of TC patients’ stratification could aid their clinical management. The material consisted of formalin-fixed paraffin-embedded tumor fragments of 49 patients who underwent thyroidectomy during the early stages of papillary TC (PTC). Tumor DNA and RNA were subjected to next-generation sequencing (NGS) on Ion Proton using the Oncomine™ Comprehensive Assay panel. We observed a significant correlation between BRAF V600E and a higher EU-TIRADS score (p-value = 0.02) with a correlation between hypoechogenicity and taller-than-wide tumor shape in analysed patients. There were no other significant associations between the identified genetic variants and other clinicopathological features. For TC patient’s stratification, a strong suspicion of BRAF V600E negativity in preoperative management of TC patients could limit the over-treatment of asymptomatic, very low-risk, indolent disease and leave room for active surveillance.

scholarly journals Specificity and sensitivity of immunohistochemical detection of factor VIII/von Willebrand factor antigen in formalin-fixed paraffin-embedded tissue.

1982 ◽  
Vol 30 (4) ◽  
pp. 371-377 ◽  
Author(s):  
R D McComb ◽  
T R Jones ◽  
S V Pizzo ◽  
D D Bigner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunohistochemical Detection ◽  
Normal Tissues ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen ◽  
Formalin Fixed

The immunohistochemical detection of factor VIII/von Willebrand factor antigen (FVIII/vWF-AG) in formalin-fixed paraffin-embedded tissues was investigated using the peroxidase-antiperoxidase (PAP) method. Highly purified human FVIII/vWF was used to raise rabbit anti-FVIII/vWF-AG serum. In addition to anti-FVIII/vWF-AG activity, the unabsorbed antiserum had anti-IgG, anti-IgM, and anti-alpha2-macroglobulin specificities. Following exhaustive absorption with these proteins, the antiserum reacted monospecifically for FVIII/vWF-AG in immunodiffusion, immunoelectrophoresis, and PAP immunohistochemistry. Sections of normal tissues from six patients and a total of 43 neoplasms were examined. Treatment of the tissue sections with trypsin prior to application of the antiserum markedly increased the sensitivity of FVIII/vWF-AG detection. The positive staining for FVIII/vWF-AG was restricted to endothelial cells in both neoplastic and nonneoplastic tissue. In general, the hyperplastic endothelia in neoplastic and reactive tissues stained more intensely than those in normal tissues. Expression of FVIII/vWF-AG by nonendothelial neoplastic cells was not observed. FVIII/vWF-AG is a reliable marker for endothelial cells.

scholarly journals The Expression of hsp-miRNA-200b-3p and -200c-3p in Human Cytomegalovirus-infected Formalin-Fixed, Paraffin-Embedded Tissues

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S358-S358
Author(s):  
Kyoung Hwa Lee ◽  
Seoyeon Min ◽  
Seul Gi Yoo ◽  
Beom Jin Lim ◽  
Jeong-Hyeon Jo ◽  
...  
Keyword(s):  
Real Time ◽  
Human Cytomegalovirus ◽  
Gene Activation ◽  
Invasive Disease ◽  
Lytic Replication ◽  
Normal Tissues ◽  
Early Protein ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Background Human cytomegalovirus (HCMV), which exist as asymptomatic latent status, can cause the tissue invasive disease through reactivation in various immunocompromised conditions. Hsp-microRNA has a specific function of post transcriptional suppression through binding with 3’ untranslated region (UTR) of mRNA. In previous study, hsp-miR-200b-3p and -200c-3p had high probability of conjugation with 3’UTR of mRNA encoded by HCMV UL 122–123 region, which translate the immediate early protein 2 (IE2) protein. IE2 (pp86) plays an essential role to initiate and regulate viral early (E) gene activation as well as propagate the subsequent steps of HCMV lytic replication. This study was aimed to evaluate whether HCMV-infected tissue had a lower expression level of hsp-miR-200b-3p and -200c-3p. Methods We had collected the formalin-fixed, paraffin-embedded tissues (FFPEs) with cytopathic pathologic findings as well as positive immunohistochemical stain (IHC) test for HCMV (N = 111). The HCMV-uninfected normal tissues (N = 77) were selected among FFPEs with neither infection nor inflammation as well as negative HCMV IHC test. We performed TaqMan® MicroRNA real-time RT-PCR to measure the expression levels of hsp-miR-200b-3p and -200c-3p and TaqMan® real-time PCR for HCMV UL83 region to measure HCMV viral load in each FFPE. We utilized the standard curves consisting of mirVanaTM miRNA mimics corresponding to each of two miRNAs, ranging from 106 to 101 copies/µL and HCMV NIBSC 09/162 strain, ranging from 5 X 106 to 5 X 101 IU/mL. Results The levels of hsp-miR-200b-3p and -200c-3p were strongly correlated with r=0.844 (P < 0.001). The expressions levels of hsp-miR-200b-3p in HCMV-infected FFPEs (log10 3.50 ± 0.13 copies/µL) were significantly lower than normal tissues (log10 5.24 ± 0.12 copies/µL of input RNA, P < 0.001). Also, HCMV-infected FFPEs were significantly lower levels of hsp-miR-200c-3p compared than normal tissues (log10 5.28 ± 0.18 vs. 7.81 ± 0.11 copies/µL of input RNA, P = 0.025). The levels of miR-200b-3p and -200c-3p had the significant inverse correlation with HCMV VL (200b-3p, spearman r=-0.392, P < 0.001 and 200c-3p, spearman r=-0.355, P < 0.001). Conclusion The low expression of hsp-miRNA-200b-3p and -200c-3p could play a pathophysiologic role of development of HCMV tissue-invasive disease. Disclosures All authors: No reported disclosures.

Comparison of DNA and RNA analyte extraction and melanin removal methods from formalin-fixed, paraffin-embedded (FFPE) melanoma.

2015 ◽  
Vol 33 (15_suppl) ◽  
pp. e20002-e20002
Author(s):  
Lauren Elaine Haydu ◽  
Tiffany L Calderone ◽  
John Miller ◽  
Roland L. Bassett ◽  
Aron Joon ◽  
...  
Keyword(s):  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed
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