Abstract:Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture micr… Show more
“…To specifically isolate normal stroma and CAS from within a section of FFPE tissue, we had previously established a workflow for case selection, tissue preparation and staining, LCM, and isolation of RNA, which can be followed by RNA analysis by either RT-qPCR or NGS (Fig. 1 ) and [ 8 ]. We used the ArcturusXT™ Laser Capture Microdissection System (Thermo Scientific) to isolate matched normal and CAS from 13 clinical cases of canine simple mammary carcinoma (Table 1 ).…”
Section: Resultsmentioning
confidence: 99%
“…[ 17 , 20 ]), we could still observe clearly macroscopically visual pieces of tissue that would not dissolve, and most likely still contained substantial amounts of RNA that remained inaccessible to our extraction efforts, contributing to low total RNA yields (Fig. 3 a; Additional file 1 : Table S1 and [ 8 ]). Fig.…”
Section: Resultsmentioning
confidence: 99%
“…All samples were formalin-fixed, paraffin-embedded tissue samples either from the Small Animal Hospital of Zurich or external cases sent in by veterinarians practising in Switzerland. Details regarding selection criteria are described in [ 8 ]. Paraffin blocks were routinely kept at room temperature.…”
Section: Methodsmentioning
confidence: 99%
“…RT-qPCR were run in duplicates on the CFX384 Touch™ Real-Time PCR detection system (BioRad). Details regarding primers can be found in [ 8 ], and other details regarding the setup of the RT-qPCR can be found in [ 11 ].…”
Section: Methodsmentioning
confidence: 99%
“…We have recently established a procedure to extract cancer-associated stroma (CAS) from archival canine FFPE breast cancer tissue by LCM using reverse-transcription quantitative PCR (RT-qPCR) [ 8 ]. During the establishment of this procedure, a major problem we encountered was the low overall yield of RNA that could be extracted from the tiny amounts of tissue that were isolated, which rendered the tissue sampling and analysis slow and cumbersome, if not almost impossible.…”
BackgroundFormalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA–protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult.MethodsWe excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step.ResultsWe successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well.ConclusionsWe present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-017-0099-7) contains supplementary material, which is available to authorized users.
“…To specifically isolate normal stroma and CAS from within a section of FFPE tissue, we had previously established a workflow for case selection, tissue preparation and staining, LCM, and isolation of RNA, which can be followed by RNA analysis by either RT-qPCR or NGS (Fig. 1 ) and [ 8 ]. We used the ArcturusXT™ Laser Capture Microdissection System (Thermo Scientific) to isolate matched normal and CAS from 13 clinical cases of canine simple mammary carcinoma (Table 1 ).…”
Section: Resultsmentioning
confidence: 99%
“…[ 17 , 20 ]), we could still observe clearly macroscopically visual pieces of tissue that would not dissolve, and most likely still contained substantial amounts of RNA that remained inaccessible to our extraction efforts, contributing to low total RNA yields (Fig. 3 a; Additional file 1 : Table S1 and [ 8 ]). Fig.…”
Section: Resultsmentioning
confidence: 99%
“…All samples were formalin-fixed, paraffin-embedded tissue samples either from the Small Animal Hospital of Zurich or external cases sent in by veterinarians practising in Switzerland. Details regarding selection criteria are described in [ 8 ]. Paraffin blocks were routinely kept at room temperature.…”
Section: Methodsmentioning
confidence: 99%
“…RT-qPCR were run in duplicates on the CFX384 Touch™ Real-Time PCR detection system (BioRad). Details regarding primers can be found in [ 8 ], and other details regarding the setup of the RT-qPCR can be found in [ 11 ].…”
Section: Methodsmentioning
confidence: 99%
“…We have recently established a procedure to extract cancer-associated stroma (CAS) from archival canine FFPE breast cancer tissue by LCM using reverse-transcription quantitative PCR (RT-qPCR) [ 8 ]. During the establishment of this procedure, a major problem we encountered was the low overall yield of RNA that could be extracted from the tiny amounts of tissue that were isolated, which rendered the tissue sampling and analysis slow and cumbersome, if not almost impossible.…”
BackgroundFormalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA–protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult.MethodsWe excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step.ResultsWe successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well.ConclusionsWe present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-017-0099-7) contains supplementary material, which is available to authorized users.
Objective
Breast cancer (BC) is a malignant tumor which threat to women's physical and mental health. However, the mechanism of metabolism alteration in BC remains unclear. This study is intended to figure out the relationship between the alternation of metabolism and the progression of BC.
Methods
In this study, metabolites of plasma in 60 BC patients and 40 healthy volunteers were detected using liquid chromatography mass spectrometer (LC‐MS). Transcriptomic data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was performed to enrich the pathways.
Results
A total of 97 metabolites have been identified and measured, of which 17 compounds exhibited the differential expression between tumor group and control group (p < 0.05; FDR < 0.05). Metabolites set enrichment analysis (MSEA) displayed that there were 12 significantly enriched pathways in all. Through the KEGG database, 382 genes were found closely correlated with the altered metabolic pathways. TCGA and GEO transcriptomic profiling revealed that 5,018 genes significantly changed between tumor group and control group. Integrating these genes with the transcriptomic data from the corresponding KEGG data set, we identified most of the differential expressed genes were related to purine metabolism. A total of 28 different expression genes were hub genes, wherein AMPD1 and RRM2 were significantly effective in the prediction of survival of BC patients, with 0.04 and 0.02, respectively.
Conclusions
Combining with the transcriptomic and metabolomics data, we found that the dysregulation of purine metabolism pathway might affect the progression of BC.
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