scholarly journals Target Enrichment Enables the Discovery of lncRNAs with Somatic Mutations or Altered Expression in Paraffin-Embedded Colorectal Cancer Samples

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2844 ◽  
Author(s):  
Susana Iraola-Guzmán ◽  
Anna Brunet-Vega ◽  
Cinta Pegueroles ◽  
Ester Saus ◽  
Hrant Hovhannisyan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transcriptional Profiling ◽  
Differentially Expressed ◽  
Specific Expression ◽  
Altered Expression ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
New Biomarkers

Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks.

scholarly journals Detection of RAF fusion transcripts in FFPE samples of Medullablastoma and Ependymom in Iraqi children with RT-RQPCR assays

2014 ◽  
Vol 11 (3) ◽  
pp. 1411-1419
Author(s):  
Baghdad Science Journal
Keyword(s):  
Brain Tumours ◽  
Large Scale ◽  
Fusion Genes ◽  
Fusion Transcripts ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Paediatric Brain Tumours ◽  
Development And Validation

Medulloblastomas and ependymomas are the most common malignant brain tumors in children. However genetic abnormalities associated with their development and prognosis remain unclear. Recently two gene fusions, KIAA1549–BRAF and SRGAP3–RAF1 have been detected in a number of brain tumours. We report here our development and validation of RT-RQPCR assays to detect various isoforms of these two fusion genes in formalin fixed paraffin embedded (FFPE) tissues of medulloblastoma and ependymoma. We examined these fusion genes in 44 paediatric brain tumours, 33 medulloblastomas and 11 ependymomas. We detected both fusion transcripts in 8/33, 5/33 SRGAP3 ex10/RAF1 ex10, and 3/33 KIAA1549 ex16/BRAF ex9, meduloblastomas but none in the 11 ependymomas examined. This investigation provided evidence to the value of RT-RQPCR assays for the detection of these fusion genes in large-scale studies on FFPE tissues. The study also reports the first detection of RAF fusion genes in meduloblstomas.

A genetic signature of relapse in stage II colorectal cancer derived from formalin fixed paraffin embedded tissue (FFPE) tissue using a unique disease specific colorectal array

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 3519-3519 ◽  
Author(s):  
P. G. Johnston ◽  
K. Mulligan ◽  
E. Kay ◽  
J. Black ◽  
S. Moore ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gene Signature ◽  
Ffpe Tissue ◽  
Stage Ii ◽  
Cancer Disease ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Stage Ii Colorectal Cancer ◽  
Disease Specific

3519 Background: We have developed the first disease specific microarray for colorectal cancer using a transcriptome-based approach. This unique tool has been specially designed and optimised for analysis of gene expression profiles from formalin fixed paraffin embedded tissues (FFPE). To evaluate this tool we are conducting a study in patients with Dukes B (stage II) colorectal cancer (CRC) using FFPE tissues to determine a prognostic genetic signature predicting disease recurrence. Methods: The colorectal array was developed using a high-throughput transcriptome-based approach and encodes over 52,500 transcripts expressed in normal and diseased colorectal tissue. To date, tumours from 32 patients have been selected (mean FFPE tissue block age - 10.4 years) from the stage II CRC cohort of a phase III randomized trial comparing 5-FU/Leucovorin versus no adjuvant treatment (NI240). The patients were from the observation alone arm;19 of whom were disease-free for 5 years post-randomisation, while 13 suffered relapse prior to 3 years. Results: RNA was extracted from all 32 FFPE tissue samples, amplified, labelled and hybridised to the colorectal cancer disease specific array. Raw data was normalised using scalar, median-polish and z-score strategies and scalar was selected as the best strategy in relation to predictive error. Within this data set genes selected by correlation using ANOVA performed best in both KNN and support vector machine algorithms (supervised). Using this approach a gene signature containing 48 genes demonstrated 100% accuracy in the prediction of relapse in stage II CRC (p<0.001). This signature was also able to separate samples at the meta-node level using unsupervised hierarchical clustering. Conclusion: We have developed the first colorectal cancer disease specific microarray and demonstrated its use using FFPE tissues. Using this approach we have derived a gene signature that predicts for a high likelihood of early relapse in stage II CRC. [Table: see text]

scholarly journals Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Suk Yee Lam ◽  
Athanasia Ioannou ◽  
Prokopis Konstanti ◽  
Thijmen Visseren ◽  
Michail Doukas ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
16S Rrna ◽  
Nested Pcr ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing ◽  
Bacterial Contaminants ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract Background Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.

scholarly journals Hubungan Lama Penyimpanan Sampel Arsip Jaringan Dalam Blok Parafin Terfiksasi Formalindengan Kualitas Hasil Ekstraksidna Mitokondria Jaringan

2018 ◽  
Vol 1 (3) ◽  
pp. 157-162
Author(s):  
Zen Hafy ◽  
Veny Larasati ◽  
Riana Sari Puspita ◽  
Novizar S ◽  
Haekal M ◽  
...  
Keyword(s):  
Storage Time ◽  
Molecular Study ◽  
Ffpe Tissue ◽  
Genetic Studies ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Pcr Products ◽  
Ffpe Tissues

Formalin-fixed paraffin-embedded (FFPE) archival tissue presents a readily available resource in molecular study nowadays. The quality of nucleid acid, especially mitoconhdrial DNA (mtDNA) extracted from FFPE tissue could be affected by the storage time. Thus, this study investigated if the FFPE tissue’s storage time had an effect on the quality of the extracted mtDNA at Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. DNA was extracted from 16 randomly selected archival FFPE tissues in Laboratory of Pathology Anatomy, RSUP dr. Mohammad Hoesin Palembang. The samples were grouped based on their storage time (less than 1 year and 1 to 5 yrs.’ old). The isolated DNA from each group was amplified using PCR with two primer pairs specifically designto amplify mtDNA of 320 bp and 142bp length, respectively.  The PCR products were visualized by electrophoresismethod. None of the samples from both groups could be amplified witht the 320bp primers. However, the PCR result of the 149 bp primers showed positive for all of the samples from each study group. The study indicated that the storage time does not affect the quality of mtDNA isolated from the FFPE samples archived in Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. Furthermore, the study showed that the mtDNA extracted from FFPE tissue has been degraded, therefore, the samples are not suitable for genetic studies require a long mtDNA amplicon.

scholarly journals A preliminary study of microRNA expression in different types of primary melanoma

2019 ◽  
Author(s):  
Ioana Gencia ◽  
Flavia Baderca ◽  
Stefania Avram ◽  
Armand Gogulescu ◽  
Anca Marcu ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Human Cancer ◽  
Primary Melanoma ◽  
Altered Expression ◽  
Mirna Expression Pattern ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
First Time ◽  
Clinical Emergency

MicroRNAs (miRNAs) have been proven to regulate the development and progression of cancer through various mechanisms. The aim of the present study was to compare miRNA expression between primary melanomas from different sites. We analyzed the expression of 84 miRNAs in 27 primary melanoma and 5 nevus formalin-fixed paraffin-embedded (FFPE) samples using the Human Cancer PathwayFinder miScript miRNA PCR Array. The FFPE samples were obtained from the archives of the Municipal Clinical Emergency Hospital of Timisoara and included 10 cutaneous melanomas, 10 uveal melanomas, 7 mucosal melanomas, and 5 cutaneous nevi. Out of 84 miRNAs, 11 miRNAs showed altered expression in all types of melanoma compared with the nevi. Among these, miR-155-5p, miR-9-5p, miR-142-5p, miR-19a-3p, miR-134-5p, and miR-301a-3p were upregulated, while miR-205-5p, miR-203a-3p, miR-27b-3p, miR-218-5p, and miR-23b-3p were downregulated. The highest similarity in miRNA expression pattern was found between uveal and mucosal melanoma groups, i.e., 15 miRNAs had altered expression in both groups. Overall, we identified several miRNAs with significantly altered expression in primary melanomas, including those reported for the first time in this type of cancer. Among them, mir-9-5p, mir-203a-3p, mir-19a-3p, mir-27b-3p, and mir-218-5p showed altered expression in all melanoma groups vs. nevus group. Further research should explore the potential of these miRNAs in melanoma.

scholarly journals Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5421
Author(s):  
Alessandra Borgognone ◽  
Garazi Serna ◽  
Marc Noguera-Julian ◽  
Lidia Alonso ◽  
Mariona Parera ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Genomics ◽  
Internal Quality ◽  
Microbial Composition ◽  
Associated Bacteria ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Ffpe Tissues ◽  
Formalin Fixed

Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses.

scholarly journals Expression of miR-127, miR-154, and miR-183 in Medullary Thyroid Carcinoma Tumors

2021 ◽  
Author(s):  
Mahsa RAHMANI SAMANI ◽  
Marjan ZARIF-YEGANEH ◽  
Atefeh MEHRABI ◽  
Amir Nader EMAMI RAZAVI ◽  
Sara SHEIKHOLESLAMI ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Rna Extraction ◽  
Control Group ◽  
Tissue Samples ◽  
Altered Expression ◽  
Medullary Thyroid ◽  
Formalin Fixed Paraffin ◽  
Tumor Tissues ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues

Background: Medullary thyroid cancer (MTC) accounts for 5%–10% of all thyroid cancers, but causes 13% of all thyroid cancer related deaths. MicroRNAs (miRs) have key functions in the development and progression of MTC. Altered expression of some miRs has been reported in many human cancers, including Thyroid cancer. Therefore, we aimed to analyze the expression of miR-154, miR-183 and miR-127 in MTC tumor tissues. Methods: In this case-control study, 15 MTC Formalin-fixed, paraffin-embedded (FFPE) tissue samples and 15 adjacent normal thyroid FFPE tissues, as a control group, were collected from Taleghani, and Loghman Hakim Hospitals, Tehran, Iran since 2005 till 2015. After RNA extraction and cDNA synthesis, the expression of miR127, miR-154 and miR-183 was measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: Our data showed a significant increase in the expression of miR-127 in MTC samples in comparison with the control group (P<0.05). Although miR-154 and miR-183 expression levels had increase expression in MTC tumors, this change was not statistically significant. Conclusion: The miR-127 could be considered as a prognostic, diagnostic and therapeutic marker for the management of MTC, and it is proposed for further investigation to fully establish the role of this miRNA in MTC.

scholarly journals Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs

2021 ◽  
pp. 104063872098688
Author(s):  
Andrea M. Camargo-Castañeda ◽  
Lauren W. Stranahan ◽  
John F. Edwards ◽  
Daniel G. Garcia-Gonzalez ◽  
Leonardo Roa ◽  
...  
Keyword(s):  
Accurate Diagnosis ◽  
Testicular Atrophy ◽  
Detection Techniques ◽  
Formalin Fixed Paraffin ◽  
Brucella Canis ◽  
Formalin Fixed Paraffin Embedded ◽  
Variable Sensitivity ◽  
Ffpe Tissues ◽  
Formalin Fixed

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term “orchitis” is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

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