mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues

Abstract Background The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. Results In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3′-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. Conclusions Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2–3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies

Proteomic patterns associated with response to breast cancer neoadjuvant treatment

Abstract Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry‐based proteomic approach and analyzed a breast cancer cohort of 113 formalin‐fixed paraffin‐embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor‐adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post‐treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER‐positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy

Impact of climate change on fishes in complex Antarctic ecosystems

Antarctic marine ecosystems are increasingly threatened by climate change and are considered to be particularly sensitive because of the adaptation of most organisms to cold and stable environmental conditions. Fishes play a central role in the Antarctic marine food web and might be affected by climate change in different ways: (i) directly by increasing water temperatures, decreasing seawater salinity and/or increasing concentrations of CO2; (ii) indirectly by alterations in the food web, in particular by changes in prey composition, and (iii) by alterations and loss of habitat due to sea ice retreat and increased ice scouring on the sea floor. Based on new data and data collected from the literature, we analyzed the vulnerability of the fish community to these threats.The potential vulnerability and acting mechanisms differ among species, developmental stages and habitats. The icefishes (family Channichthyidae) are one group that are especially vulnerable to a changing South Polar Sea, as are the pelagic shoal fish species Pleuragramma antarcticum. Both will almost certainly be negatively affected by abiotic alterations and changes in food web structure associated with climate change, the latter additionally by habitat loss. The major bottleneck for the persistence of the majority of populations appears to be the survival of early developmental stages, which are apparently highly sensitive to many types of alterations. In the long term, if climate projections are realized, species loss seems inevitable: within the demersal fish community, the loss or decline of one species might be compensated by others, whereas the pelagic fish community in contrast is extremely poor in species and dominated by P. antarcticum. The loss of this key species could therefore have especially severe consequences for food web structure and the functioning of the entire ecosystem.Fil: Mintenbeck, Katja. Alfred Wegener Institute For Polar And Marine Research; AlemaniaFil: Barrera Oro, Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; ArgentinaFil: Brey, Thomas. Alfred Wegener Institute For Polar And Marine Research; AlemaniaFil: Jacob, Ute. Universitat Hamburg; AlemaniaFil: Knust, Rainer. Alfred Wegener Institute For Polar And Marine Research; AlemaniaFil: Mark, Felix C.. Alfred Wegener Institute For Polar And Marine Research; AlemaniaFil: Moreira, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; ArgentinaFil: Strobel, Anneli. Alfred Wegener Institute For Polar And Marine Research; AlemaniaFil: Arntz, Wolf E.. Alfred Wegener Institute For Polar And Marine Research; Alemani

Recession, employment and self-rated health: a study on the gender gap

Objectives: Employment status and economic recession have been associated with negative effects on self-rated health, and this effect differs by gender. We analysed the effects of the Spanish economic recession in terms of self-rated health, its differential effect among genders and its influence on gender gap. Study design: Repeated cross-sectional study using Spanish health surveys (2001–2014). Methods: Logistic regression models were conducted to explore the association between self-rated health and employment status and its evolution over time and gender. To test the impact of the economic recession, pooled data regression models were conducted. Results: In this study, we considered 104,577 subjects. During the last 15 years, women have entered the labour market, leading to wide changes in the Spanish traditional family roles. Instead of an increasing proportion of women workers, gender employment differences persist. Therefore, in 2014, the prevalence of workers was 55.77% in men, whereas in women, it was 44.01%. Self-rated health trends during the economic recession differ by gender, with women improving slightly their self-rated health from a low self-rated health prevalence of 38.76% in 2001 to 33.78% in 2014. On the contrary, men seem more vulnerable to employment circumstances, which have led to substantial reduction in the gender gap. Conclusions: Although a gender gap persists, the change in socio-economic roles seems to increase women's self-rated health, reducing this gap. It is important to promote women's labour market inclusion, even in economic recession periods

Additional file 1: of mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues

Table S1. RNA integrity number for all samples. (PDF 212 kb

Additional file 5: of mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues

Figure S2. Comparison of fold-changes measured for FFPE samples vs. matched FF samples using mRNA-seq. (A) Scatter plot for the expression fold changes (log2 scale) of genes measured in T1 vs.T3, obtained from FF samples (x-axis) compared to matched FFPE samples (y-axis) by mRNA-seq protocol (purple). r-square and correlation coefficient are presented at the plot. B) Scatter plot for the expression fold changes (log2 scale) of genes measured in T2 vs.T3, obtained from FF samples (x-axis) compared to matched FFPE samples (y-axis) by mRNA-seq protocol (purple). r-square and correlation coefficient are presented at the plot. (PDF 936 kb

Additional file 6: of mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues

Figure S3. Expression of non-coding RNAs in FFPE samples by mRNAseq and RiboZero protocols. (A) Scatter plot of the expression levels of annotated lincRNAs as measured on T1 FFPE sample by mRNAseq (x-axis) versus RiboZero protocol (y-axis). Correlation coefficients between the two protocols for the expression of these lincRNAs are indicated at the fig. (B) Same as (A) for miRNAs expression. (PDF 185 kb