Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA137377 | ENA |
REPOSITORIES: ENA
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Project description:INTRODUCTION. Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. RESULTS. The cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues. Thirty samples from human cancers processed in different ways before RNA extraction: (i) Fresh-Freezing (FF), (ii) Standard Fixation with formalin (SF), (iii) Cold-Fixation (CF), a new formalin fixation procedure preserving nucleic acids. The first six samples compare CF with SF and FF in one colorectal cancer (CRC) specimen and one breast cancer (BRCa) specimen. The subsequent 24 samples compare only CF with FF in various cancer specimens. Expression data are neither normalized nor backound subtracted to preserve the original signal distribution for each sample, which is essential to compare expression profiles obtained after different tissue preservation methods.
2011-09-04 | E-GEOD-27175 | biostudies-arrayexpress
Project description:INTRODUCTION. Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. RESULTS. The cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.
2011-09-05 | GSE27175 | GEO
Project description:<p>The Biospecimen Pre-analytical Variables (BPV) Program is a National Cancer Institute-sponsored study to systematically assess the effects of pre-analytical factors on the molecular profile of biospecimens. A robust biospecimen collection infrastructure was established to prospectively collect biospecimens using rigorous standard operating procedures to control for most variables while introducing experimental conditions to study specific biospecimen handling issues, including the cold ischemic time (delay to formalin fixation), time in formalin, freezing methods, and storage temperatures and durations. RNA and DNA from biospecimens collected under these conditions was analyzed on multiple molecular platforms. The potential effects of these pre-analytical conditions on protein integrity and detection of metabolites were also examined. Data from this study will be used to develop evidence-based biospecimen standard operating procedures and best practices for fit-for-purpose collection, processing, and storage of biospecimens.</p> <p>The BPV Cohort is utilized in the following dbGaP substudies. To view genotypes, analysis, expression data, other molecular data, and derived variables collected in these substudies, please click on the following sub-studies below or in the "Substudies" box located on the right hand side of this top-level study page phs001304 BPV Cohort. The substudy links will be active once they are released by dbGaP.</p> <p> <ol> <li>Preanalytical Impacts on Global Metabolite Profiling - plasma (MassSpec by Metabolon) This study was to evaluate the impact of the storage temperature (s) (-80°C and LN2 vapor) and the length of storage on human plasma quality using LC-MS/MS (liquid-chromatography-mass spectrometry/mass spectrometry) based global metabolite profiling. The study includes 240 plasma samples collected from 40 donors.</li> <li>Investigate the effect of the delay to fixation on the proteome and phosphoproteome -FFPE (MassSpec by Caprion). The study is to do proteome and phosphoproteome analysis on Delay to fixation was carried out using FFPE tumor samples from colon and ovarian cancer patients comparing 2, 3, and 12hr delay to fixation to the 1hr time point. The study includes 100 samples 20 donors.</li> <li>Investigate the effect of storage conditions of tumor specimens on the proteome and phosphoproteome profiling- Frozen tissue and plasma (MassSpec by Caprion). The study was to evaluate the effects of storage conditions on tumor specimens. Plasma samples from 40 cancer patients stored at two different temperatures (-80°C and LN2) for a given period (0-2, 6-8, and 12-14 months) were evaluated. Frozen kidney tumor samples from 20 patients were compared for effects of different snap frozen (dry ice vs. LN2) and storage temperatures (-80°C and LN2). The study includes 100 tissue and 240 plasma samples from 60 donors.</li> <li>Preanalytical Impacts on Genomic Sequencing by Next Generation Sequencing (NGS) technology (mRNA/miRNA and WES by Expression Analysis). The goal of the study is to determine the effects of cold ischemic delay-to-fixation (4 time points) and formalin preservation (FFPE) on the nature and quality of genomic profiles using the matched freshly frozen sample as the gold standard, which including WES, RNAseq. The study includes 395 samples from 37 donors.</li> <li>Preanalytical Impacts on Copy Number Variation (CNV) Detection by aCGH technology (aCGH by Georgetown University). This study was to use aCGH to evaluate the effect of variation in cold ischemia time and time in formalin fixation on CNV in DNA extracted from kidney cancer specimens. The study includes 235 samples from 40 donors.</li> <li>Evaluation of frozen conditions on mRNA profiling by TaqMan assay (mRNA expression by Georgetown University). This study was to utilize gene expression profiling, using custom TaqMan arrays, to compare the molecular profiles of RNA from frozen tumor samples collected using two freezing methods (dry ice or LN2), two storage temperatures (-80°C or LN2 vapor), as well as Optimal Cutting Temperature (OCT) compound and non-OCT embedded. The study includes 100 samples from 20 donors.</li> <li>mRNA signature for stratification by cold ischemia time (mRNA expression by IBBL). The study was to determine the effects of cold ischemic time (delay-to-fixation) and formalin preservation (FFPE) on mRNA detection by Taqman assay using tumor tissue specimens from kidney, colon and ovarian cancer patients. There are160 samples from 40 donors.</li> </ol> </p> <p><b>The Biospecimen PV cohort is utilized in the following dbGaP individual studies.</b> To view molecular data, and derived variables collected in these individual studies, please click on the following individual studies below or in the "Sub-studies" box located on the right hand side of this top-level study page <a href="study.cgi?study_id=phs001304">phs001304</a> Biospecimen PV cohort. <ul> <li><a href="study.cgi?study_id=phs001634">phs001634</a> CIT mRNA</li> <li><a href="study.cgi?study_id=phs001635">phs001635</a> CNV aCGH</li> <li><a href="study.cgi?study_id=phs001636">phs001636</a> Fixation Delay</li> <li><a href="study.cgi?study_id=phs001637">phs001637</a> Global Metabolite Profiling</li> <li><a href="study.cgi?study_id=phs001638">phs001638</a> mRNA TaqMan</li> <li><a href="study.cgi?study_id=phs001639">phs001639</a> NGS</li> <li><a href="study.cgi?study_id=phs001640">phs001640</a> Tumor Storage</li> </ul> </p>
| phs001304 | dbGaP
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
2020-09-08 | GSE148174 | 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:Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin embedded (FFPE) tissue samples. Due to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromised, meaning obtaining informative data regarding epigenetic, genomic and expression alterations can be challenging. Here we have investigated the utility of repairing damaged DNA derived from FFPE tumours prior to single nucleotide polymorphism (SNP) arrays for whole genome DNA copy number analysis.
2013-07-01 | GSE43406 | GEO
Project description:Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we first sought to understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin for 15 minutes, 1 hour, 6 hours, and 24 hours. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and enables semi-quantitative estimates of relative gene expression in this yeast model.
2024-03-01 | GSE256156 | GEO
Project description:Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we first sought to understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin for 15 minutes, 1 hour, 6 hours, and 24 hours. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and enables semi-quantitative estimates of relative gene expression in this yeast model.
2024-03-01 | GSE256160 | GEO
Project description:Formalin-fixed paraffin embedded (FFPE) is the clinical gold standard of tissue preservation globally. We present a novel method for optimal extraction of proteins from FFPE tissue samples. The method combines non-toxic, proteomic inert mineral oil for deparaffinization, followed by extraction using elevated temperature and pressure (121˚C; 15 psi). Our novel TOP (Temperature, Oil, and Pressure) proteome extraction is four times faster, less laborious and increases the protein yield. Furthermore, the TOP method is environmentally friendly, as opposed to traditional methods. Comparison of extracted protein by mass spectrometry based proteomics revealed that TOP is not sensitive to formalin over-fixation, unlike the traditional approach, a fact that makes it suitable in the clinical setting. We expect that the TOP proteome extraction platform will help to alleviate some of the difficulties proteomics is facing in the clinical setting.
2024-02-12 | PXD002526 | Pride
Project description:To identify tumor compartment-specific microRNA expression in stage I non-small cell lung cancer in humans, surgically resected and formalin-fixed tumor tissues were used in laser capture microdissection to isolate tumor epithelia and stroma. Total RNA extracted from the microdissectates was analyzed for microRNA expression using the 7th generation miRCURY™ locked nucleic acid microarray platform (Exiqon®, Vedbaek, Denmark).
2014-12-31 | GSE42425 | GEO