Dataset Information

Comparative proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

ABSTRACT: Clinical proteomics research is in part limited by the availability of clinical samples. However, large biobanks exist worldwide containing formalin-fixed and paraffin embedded samples and samples stored in RNAlater. The extraction of proteins for proteome analysis from samples prepared by either method has been demonstrated. However, the impact of the preservation method on the result of a quantitative proteome analysis remains largely uninvestigated. We, therefore, conducted a proteome analysis of human colon mucosal biopsies where the material had been preserved accordingly. Twenty-four colon mucosal biopsies were extracted from the sigmoideum by endoscopy from two participants. The biopsies were either directly-frozen, stabilized in RNAlater, or stabilized by formaldehyde fixation immediately or incubated for 30 min to simulate a clinical situation, followed by paraffin embedding. The impact of the preservation method on the result of a proteome analysis was characterized by high throughput gel free quantitative proteomics.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colonic Mucosa, Colon Sigmoideum

DISEASE(S): Disease Free

SUBMITTER: Tue Bjerg Bennike

LAB HEAD: Allan Stensballe

PROVIDER: PXD002029 | Pride | 2015-11-23

REPOSITORIES: Pride

ACCESS DATA

Publications

Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies.

Bennike Tue Bjerg TB Kastaniegaard Kenneth K Padurariu Simona S Gaihede Michael M Birkelund Svend S Andersen Vibeke V Stensballe Allan A

Data in brief 20160206

Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving samples in RNAlater, and by formalin-fixation, paraffin-embedding on human soft tissue, using directly frozen samples as a control ("Comparing the proteome of snap frozen, RNAlater preserved, and formal ...[more]

PMID: 26937473

Similar Datasets

Project description:Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlaterÂ® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients, to enable the decision of whether or not it is feasible to apply RNAseq technology to archival tissue. Core biopsies were obtained with a 16g needle from 16 patients undergoing partial or full nephrectomy. The RNA sequencing libraries were generated applying the new Illumina TruSeqÂ® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlaterÂ® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n=1106) differentially expressed genes. The average expression of these transcripts in both datasets correlated with R2=0.97, and the log2 fold changes of these transcripts, which are significantly altered in both datasets, correlated with R2=0.96. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-Ã1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens. *** Access to raw data can be requested from submitters ***

Project description:CONTEXT Slowly progressive chronic tubulo-interstitial damage jeopardizes long-term renal allograft survival. Both immune and non-immune mechanisms are thought to contribute, but the most promising targets for timely intervention have not been identified. OBJECTIVE In the current study we seek to determine the driving force behind progressive histological damage of renal allografts, without the interference of donor pathology, delayed graft function and acute graft rejection. DESIGN We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. PATIENTS Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection. RESULTS In this highly cross-validated study, we demonstrate the significant association of established, ongoing and future chronic histological damage with regulation of adaptive immune gene expression (T-cell and B-cell transcript sets) and innate immune response gene expression (dendritic cell, NK-cell, mast cell and granulocyte transcripts). We demonstrate the ability of gene expression analysis to perform as a quantitative marker for ongoing inflammation with a wide dynamic range: from subtle subhistological inflammation prior to development of chronic damage, over moderate subclinical inflammation associated with chronic histological damage, to marked inflammation of Banff-grade acute T-cell mediated rejection. CONCLUSION Progressive chronic histological damage after kidney transplantation is associated with significant regulation of both innate and adaptive immune responses, months before the histological lesions appear. This study therefore corroborates the hypothesis that quantitative inflammation below the diagnostic threshold of classic T-cell or antibody-mediated rejection is associated with early subclinical stages of progressive renal allograft damage. We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection.

Project description:To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately. Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).

Dataset Information

Comparative proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

Publications

Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure