Optimizing RNA Extraction Methods for High Throughput Transcriptome Sequencing of Fresh Frozen and Formalin-Fixed Paraffin-Embedded Cardiac Tissues Specimens
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ABSTRACT:
PROVIDER: PRJNA1112367 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29058073_1.fastq.gz | Fastqsanger.gz | |||
| SRR29058073_2.fastq.gz | Fastqsanger.gz | |||
| SRR29058074_1.fastq.gz | Fastqsanger.gz | |||
| SRR29058074_2.fastq.gz | Fastqsanger.gz | |||
| SRR29058075_1.fastq.gz | Fastqsanger.gz |
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Project description:In this study, the efficiency of four RNA extraction methods was compared on 23 FFPE cardiac tissue specimens. The Qiagen AllPrep DNA/RNA FFPE kit (Method QP), Qiagen AllPrep DNA/RNA FFPE kit, with protocol modification on the ethanol wash step after deparaffinization (Method QE), CELLDATA RNAstorm 2.0 FFPE RNA Extraction kit (Method BP) and CELLDATA RNAstorm 2.0 FFPE RNA Extraction Kit with protocol modifications on the lysis step (Method BL). In comparing RNA quality metrics across FFPE RNA extract, nucleic acids extracted with Method QE and QP had the highest RNA yield. However, Method QE outperformed Method QP as more extract from Method QE had DV 200 values above 30%. Both method BL and BP produced a similar range of RNA purity and yield but more extract from Method BL had DV 200 values above 30% compared to Method BP. When accessing distribution value, Method BL outperformed Methods BP, QE, and QP as more extracts from Method BL had DV 200 values above 30% (16/23 samples) compared to other methods (PDV200<0.001; Kruskal-Wallis). However, method QE outperformed other methods in terms of RNA yield. The sequencing performance of RNA extracts from Method QE and Method BL was further tested on 8 matching samples with high RNA yield and high DV200 value respectively. RNA extracts from Method QE which yielded the highest RNA quantity among all methods exhibited comparable sequencing performance to extract obtained through Method BL, which yielded extract with high DV200 value. This study suggests that the DV200 and RNA yield are both reliable pre-analytic metrics in determining a suitable method for successful transcriptome sequencing of FFPE samples and have important implications for future studies exploring transcriptome sequencing of FFPE cardiac specimens.
2024-05-23 | GSE267659 | GEO
Project description:Archived FFPE cardiac tissue specimens are valuable for molecular studies aimed at identifying biomarkers linked to mortality in cardiovascular disease. Establishing a reliable and reproducible RNA extraction method is critical for generating high-quality transcriptome sequences for molecular assays. Here, the efficiency of four RNA extraction methods: Qiagen AllPrep DNA/RNA method (Method QP); Qiagen AllPrep DNA/RNA method with protocol modification on the ethanol wash step after deparaffinization (Method QE); CELLDATA RNA extraction (Method BP) and CELLDATA RNA extraction with protocol modifications on the lysis step (Method BL) was compared on 23 matching FFPE cardiac tissue specimens (n = 92).In comparing RNA quality metrics across FFPE RNA extract, nucleic acids extracted deploying Method QE and QP produced the highest RNA yield. However, Method QE outperformed Method QP as more extract from Method QE had DV 200 values above 30%. Both method BL and BP produced similar range of RNA purity and yield but more extract from Method BL had DV 200 values above 30% compared to Method BP. When accessing distribution value, Method BL outperformed Methods BP, QE, and QP as more extracts from Method BL had DV 200 values above 30% compared to other methods (PDV200<0.001; Kruskal-Wallis). Method QE outperformed other methods in terms of RNA yield. RNA extracts from Method QE, characterized by high RNA yield, achieved sequencing results comparable to those from Method BL, characterized by high DV200 values. Our findings reveal that optimizing protocols can yield higher-quality RNA, facilitating the exploration of more disease conditions with high-resolution transcriptome profiling.
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Project description:Both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratory-based method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies.
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Project description:We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.
| S-EPMC4651363 | biostudies-literature
Project description:Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
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Project description:BackgroundRetrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens.Principal findingsFor effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses.SignificanceWe determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.
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2024-06-20 | GSE270364 | GEO
Project description:DNA methylation is the most studied epigenetic modification due to its role in regulating gene expression, and its involvement in the pathogenesis of cancer and several diseases upon aberrations in methylation. The method of choice to evaluate genome-wide methylation has been the Illumina HumanMethylation450 BeadChip (450K), but it was recently replaced with the MethylationEPIC BeadChip (EPIC). We therefore sought to validate the EPIC array in comparison to the 450K array for both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumours. We also performed analysis on the EPIC array with paired FF and FFPE samples to adapt to a clinical setting where FFPE is routinely used. Further, we compared two restoration methods, REPLI-g and Infinium, for FFPE-derived DNA on the EPIC array. The Pearson correlation of β values for common probes on the 450K and EPIC array was high for both FF (mean: 0.992) and FFPE (mean: 0.984) samples. The β values generated from the EPIC array for FFPE samples correlated well with the paired FF tumours, but varied between 0.901 and 0.987. We did note that sample pairs with lower correlation had less bimodal density distributions of β values and displayed higher noise in the copy number alteration plots (generated from the methylation array data) in the FFPE sample. Both REPLI-g and the Infinium restoration for FFPE samples performed well on the EPIC array and generated equivalent correlation scores to the paired FF sample.
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