MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content.
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ABSTRACT:
SUBMITTER: Kakimoto Y
PROVIDER: S-EPMC5029930 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
Publications
MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content.
Kakimoto Yu Y Tanaka Masayuki M Kamiguchi Hiroshi H Ochiai Eriko E Osawa Motoki M
PloS one 20160920 9
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, ...[more]
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Project description:BackgroundAs degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation.MethodsBladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without.ResultsRNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72).ConclusionSample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.
| S-EPMC3419950 | biostudies-literature
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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.
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Project description:DNA:RNA hybrid duplex plays important roles in various biological processes. Both its structural stability and interactions with proteins are highly sequence dependent. In this study, we utilize homebuilt optical tweezers to investigate how GC contents in the sequence influence the structural and mechanical properties of DNA:RNA hybrid by measuring its contour length, elasticities, and overstretching dynamics. Our results support that the DNA:RNA hybrid adopts a conformation between the A- and B-form helix, and the GC content does not affect its structural and elastic parameters obviously when varying from 40 to 60% before the overstretching transition of DNA:RNA hybrid occurs. In the overstretching transition, however, our study unravels significant heterogeneity and strong sequence dependence, suggesting the presence of a highly dynamic competition between the two processes, namely the S-form duplex formation (nonhysteretic) and the unpeeling (hysteretic). Analyzing the components left in DNA:RNA hybrid after the overstretching transition suggests that the RNA strand is more easily unpeeled than the DNA strand, whereas an increase in the GC content from 40 to 60% can significantly reduce unpeeling. Large hysteresis is observed between the stretching and relaxation processes, which is also quantitatively correlated with the percentage of unpeeling in the DNA:RNA duplex. Increasing in both the salt concentration and GC content can effectively reduce the hysteresis with the latter being more significant. Together, our study reveals that the mechanical properties of DNA:RNA hybrid duplexes are significantly different from double-stranded DNA and double-stranded RNA, and its overstretching behavior is highly sequence dependent. These results should be taken into account in the future studies on DNA:RNA-hybrid-related functional structures and motor proteins.
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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.
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Project description:BackgroundLong-term storage of tissue slides has been reported to induce reduced biomarker (e.g. proteins and messenger RNA) detection. This study aimed to evaluate the impact of long-term storage time (0, 16, 24 and 36 weeks) and treatment conditions (non-paraffin and paraffin dipping) at 4°C on RNA quality in formalin-fixed and paraffin-embedded (FFPE) tissue sections.Materials and methodsNanoString GeoMx Digital Spatial Profiling (DSP), a novel platform that allows spatial profiling, was used to profile RNA in human bladder cancer FFPE tissue sections.ResultsWe observed excellent consistency of quantitative DSP RNA counts of all targets between two different treatment conditions (R > 0.97, Pearson correlation) at each time point and among all four different storage time points (R > 0.96, Pearson correlation) within each treatment condition. No significant difference was observed in the percentage of target genes with sufficient signal across two different treatment conditions at any time point (0 week, P = 0.96; 16 weeks, P = 0.76; 24 weeks, P = 0.96; 36 weeks, P = 0.76, Kolmogorov-Smirnov test) and across all four different storage time points (P > 0.05, Kolmogorov-Smirnov test) in either treatment condition.ConclusionAlthough both treatment conditions provided similar results in terms of count reproducibility and signal preservation, we recommend paraffin dipping to generate reproducible RNA results and optimize sample storage. Technology behind the NanoString GeoMx DSP platform shows a robust and reproducible RNA signal from multiple targets in the FFPE tissue sections stored at 4°C for at least up to 36 weeks.
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Project description:BackgroundArchived 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.ResultsAs 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.ConclusionsThrough 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:Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits ability to expression profile, we explored factors predicting success for FFPE profiling and investigated an approach overcoming this limitation. Methods: Bladder (n=141, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon1.0ST arrays. Percent detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. Precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Eight-old and -young cervix samples were compared using Affymetrix miRNA 2.0 arrays. For comparison, the 'cervix_tumour_cs1_113' (previsously submitted GSM677307) was included and re-analyzed with the samples from the current study (total 161 Cervix samples). Results: RNA quality controls (e.g. RNA integrity number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R2=-0.30, p<0.01) and cervix (R2=-0.69, p<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; p=1.10x10-5). Median miRNA probeset expression of eight-old and eight-young cervix samples correlated well (R2=0.95) overcoming the age-related bias of mRNA probesets, suggesting miR-205 stability generalises across microRNA. Conclusions: microRNA profiling overcomes the limitation of degraded FFPE samples
2012-07-04 | GSE39016 | GEO