Evaluation of capture and amplicon-based targeted sequencing methods on formalin-fixed tumours
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PROVIDER: EGAS00001004965 | EGA |
REPOSITORIES: EGA
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Project description:Three capture (Agilent’s SureSelectXT HS, Illumina’s Nextera Rapid Capture Custom, and New England Biolabs’ Next Direct Custom) and one amplicon-based (Qiagen’s Human Breast Cancer Panel) targeted sequencing methods on 6-8 paired blood and FFPE from the Malaysian Breast Cancer Cohort.
| EGAD00001006879 | EGA
Project description:Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.
| S-EPMC4444292 | biostudies-literature
Project description:Sixth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile in duplicate the expression of microRNAs in 250 or 400 ng of RNA isolated from a laser microdissectate of a formalin-fixed and paraffin-embedded human non-small cell lung cancer tumor. Laser microdissection was performed on a hematoxylin-eosin-stained, 8 um-thick section of formalin-fixed and paraffin-embedded tissue. The microdissectate was treated with proteinase K overnight at 55 ºC. Total RNA from the lysate was then extracted using FFPE RNA Isolation Kit from Norgen Biotek® (Thorold, Canada). RiboGreen assay was used to quantify RNA in the preparation. Two-hundred-fifty or 400 ng of RNA was used in duplicate for dual-channel microarray-based microRNA expression profiling. Sample RNA was labeled with the Cy3-like Hy3™ dye, mixed with a human universal reference RNA (product number AM6000, Ambion®, Austin, TX) labeled with the Cy5-like Hy5™ dye, and hybridized to the two-color miRCURY™ arrays.
2011-09-07 | E-GEOD-31946 | biostudies-arrayexpress
Project description:Method to perform spatial analysis of mRNA in FFPE and PFA fixed tissue sections using spatially barcoded slides and based on oligo(dT) mRNA capture.
2021-10-15 | GSE185715 | GEO
Project description:Sixth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile in duplicate the expression of microRNAs in 250 or 400 ng of RNA isolated from a laser microdissectate of a formalin-fixed and paraffin-embedded human non-small cell lung cancer tumor.
2011-09-08 | GSE31946 | GEO
Project description:Formalin-fixed lung or trachea tissue specimens from four infants and one adolescent who died of respiratory illness were tested for Bordetella pertussis by conventional and real-time PCR assays. B. pertussis was confirmed in all cases. PCR can be an invaluable retrospective diagnostic tool for evaluating archival tissues from patients with suspected fatal pertussis.
| S-EPMC1393166 | biostudies-literature
Project description:MicroRNAs are useful biomarkers for various disease states, and their preservation in formalin-fixed, paraffin-embedded (FFPE) tissue makes them particularly useful for clinicogenetic studies. Although global microRNA expression in FFPE samples is routinely measured with microarrays, the utility of RNA sequencing for such profiling has yet to be established. In this study, to appraise the suitability of RNA sequencing, microRNAs in RNA from pathologic stage I lung adenocarcinoma FFPE samples were quantified with 8x60K Agilent® SurePrint™ G3 Human miRNA 8x60k (release 16.0) microarray and Illumina® HiSeq™ 2000 sequencing platforms.
2014-12-31 | GSE57835 | GEO
Project description:There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across 10 archived uterine mesenchymal tumor tissue blocks, including 9 uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long-term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, mass spectrometry-based proteome profiling enables global identification and quantification of thousands of proteins without a priori knowledge of individual proteins being analyzed or the need of validated antibodies.
| S-EPMC2664629 | biostudies-literature
Project description:BackgroundProteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.ResultsThe best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.ConclusionsIn spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.
| S-EPMC3123619 | biostudies-literature
Project description:Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.
| S-EPMC6310091 | biostudies-literature