Protein Extraction From FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques.
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ABSTRACT:
SUBMITTER: Garcia-Vence M
PROVIDER: S-EPMC8158658 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The vast majority of tissue biopsies from patients in hospital environments are included in paraffin (FFPE- formalin-fixed and paraffin-embedded) for long-term storage. This fixation process produces a modification in the proteins called "crosslinks", which improves protein stability, necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical techniques, since these modifications do not suppose a drawback for this technique; however, crosslinks makes the protein extraction process a challenging one. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. This work aims to establish a deparaffinization and protein extraction protocol from paraffin-embedded kidney samples, which finally allows us to obtain high quantity and quality protein, suitable for the application of proteomic techniques. To achieve this; we have designed different protocols using a range of buffers with diverse compositions and characteristics. Our results showed that xylene can eliminate the formalin fixation from the kidney tissue. Moreover, strong detergents (SDS-sodium dodecil sulfate- or analogues) and reducing agents (e.g. DTT- Dithiothreitol-) in the extraction buffer appear to be more important to obtain a good protein extraction yields and a good number of proteins identifications by LC-MSMS.
2021-09-10 | PXD023891 | Pride
Project description:BACKGROUND:Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. RESULTS:As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<?5%), and collectively shared a "C?>?T|G?>?A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. CONCLUSIONS:Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.
| S-EPMC6720378 | biostudies-literature
Project description:MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 <r < 0.92). However, the relative read count of each miRNA was different depending on the GC content (p<0.0001). The unequal degradation of each miRNA affected the abundance ranking in the library, and miR-133a was shown to be the most abundant in FFPE cardiac tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001). We showed that deep sequencing data obtained using FFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples.
| S-EPMC5029930 | biostudies-literature
Project description:Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here, we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh-frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between 1 and 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, that is, punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts, we identified myeloperoxidase, a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.
| S-EPMC6822243 | biostudies-literature
Project description:Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.
| S-EPMC5957415 | biostudies-literature
Project description:Among women, ovarian cancer (OC) is one of the most severe forms of malignancy, accounting for a low 5-year survival rate, of approximately 52%. Early symptoms are unspecific and hence hard to detect. The origin of OC and its subtypes are still unclear, underlying the need for efficient diagnostic biomarkers. In that regard, epigenetics studies are emerging in cancer diagnostics, with encouraging outcomes. Among them, DNA methylation profiling has shown that the origins of the cancer epigenome are associated with molecular factors that are crucial to carcinogenesis, such as regulation of oncogenes and tumor suppressors. Furthermore, those events have been detected in abnormal cell morphology before neoplastic formation, indicating its potential crucial use in the OC diagnostics in the future. Nonetheless, studies are limited, and whether methylation analysis can be performed optimally in formalin-fixed paraffin-embedded (FFPE) preparations of OC cases is still elusive. In the present report, we investigated the performance of DNA methylation analysis in FFPE samples, compared to their matched fresh frozen tissue in a small cohort of OC samples. We found that the overall DNA methylation profile in FFPE tissue showed high concordance to that found in fresh frozen tissue, and accounting for the small cohort size, the differentially methylated sites found primarily in frozen tissue, compared to benign samples, were also reproducible in FFPE. Overall, by using samples from our current clinical setting of tissue preservation, these preliminary observations might provide insights into the clinical use of FFPE tissues in methylation studies without critically compromising the outcome.
| S-EPMC8526488 | biostudies-literature
Project description:Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies.
| S-EPMC5480995 | biostudies-literature
Project description:BackgroundThe surveillance and therapy of early-stage cancer would be better for patients' prognosis. However, the extreme trace amount of tissue samples in different stages have limited in portraying the characterization of early-stage cancer. Therefore, we focused on and presented comprehensive proteomic and phosphoproproteomic profiling of the trace FFPE samples from early-stage gastrointestinal cancer, and then explored the potential biomarkers of early-stage gastrointestinal cancer.MethodsIn this study, a quantitative proteomic method with chromatography with mass spectrometry (LC-MS/MS) was used to analyse the proteomic difference between the trace early-stage esophageal squamous cell carcinoma (EESCC) and early-stage duodenum adenocarcinoma cancer (EDAC).ResultsWe identified ~ 6000 proteins and > 10,000 phosphosites in single trace FFPE samples. Comparative analysis disclosed the diverse proteomic features of tumor tissues compared with paired normal tissue of EESCC and EDAC, and revealed the difference of EESCC and EDAC was derived from their origin normal tissue. The distinct separation of EESCC and EDAC illustrated the functions of cell cycle (RB1 T373, EGFR T693) in EESCC, and the positive impacts of apoptosis, metabolic processes (MTOR and MTOR S1261) in EDAC. Furthermore, we deconvoluted the immune infiltration of early-stage gastrointestinal cancer, in which higher immune cell signatures were detected in EDAC, and showed the specific cytokines in EESCC and EDAC. We performed kinases-substates relationship analysis and elucidated the specific proteomic kinase characterization of EESCC and EDAC, and proposed the medicative effects and corresponding drugs for EESCC and EDAC at the clinic.ConclusionWe disclosed the specific immune characterization of the early-stage gastrointestinal cancer, and presented potential makers of EESCC (EGFR, PDGFRB, CDK4, WEE1) and EDAC (MTOR, MAP2K1, MAPK3). This study represents a major stepping stone towards investigating the carcinogenesis mechanism of gastrointestinal cancer, and providing a rich resource for medicative strategy in the clinic.
| S-EPMC8994365 | biostudies-literature
Project description:ObjectivesFormalin-fixed paraffin-embedded (FFPE) tissue is the standard material for diagnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction protocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of functionally relevant proteins.MethodsUsing the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC-MS).ResultsCommercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval.ConclusionsPreanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.
| S-EPMC9063121 | biostudies-literature
Project description:Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE samples. In this study, to evaluate the gene expression of frozen tissue-derived prognostic signatures in FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 FFPE CRC samples measured by both platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR < 0.05) and gene set (four of the six reported multi-gene signatures with sufficient information for evaluation, P < 0.05) expression differences associated with survival outcomes. Using nCounter platform-derived data, one of the six multi-gene signatures (P < 0.05) but no individual gene was associated with survival outcomes. Our study indicated that sufficiently high quality RNA could be obtained from FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients.
| S-EPMC5021945 | biostudies-literature