Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.

Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody-based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to ∼80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.

Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer.

Ductal carcinoma in situ (DCIS) is a non-invasive form of breast cancer where cells restricted to the ducts exhibit an atypical phenotype. Some DCIS lesions are believed to rapidly transit to invasive ductal carcinomas (IDCs), while others remain unchanged. Existing classification systems for DCIS fail to identify those lesions that transit to IDC. We studied gene expression patterns of 31 pure DCIS, 36 pure invasive cancers and 42 cases of mixed diagnosis (invasive cancer with an in situ component) using Agilent Whole Human Genome Oligo Microarrays 44k. Six normal breast tissue samples were also included as controls. qRT-PCR was used for validation. All DCIS and invasive samples could be classified into the "intrinsic" molecular subtypes defined for invasive breast cancer. Hierarchical clustering establishes that samples group by intrinsic subtype, and not by diagnosis. We observed heterogeneity in the transcriptomes among DCIS of high histological grade and identified a distinct subgroup containing seven of the 31 DCIS samples with gene expression characteristics more similar to advanced tumours. A set of genes independent of grade, ER-status and HER2-status was identified by logistic regression that univariately classified a sample as belonging to this distinct DCIS subgroup. qRT-PCR of single markers clearly separated this DCIS subgroup from the other DCIS, and contains samples from several histopathological and intrinsic molecular subtypes. The genes that differentiate between these two types of DCIS suggest several processes related to the re-organisation of the microenvironment. This raises interesting possibilities for identification of DCIS lesions both with and without invasive characteristics, which potentially could be used in clinical assessment of a woman's risk of progression, and lead to improved management that would avoid the current over- and under-treatment of patients.

Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue.

Biobanks of fresh, unfixed human tissue represent a valuable source for gene expression analysis in translational research and molecular pathology. The aim of this study was to evaluate the impact of thawing on RNA integrity and gene expression in fresh frozen tissue specimens. Portions of snap frozen tonsil tissue, unfixed or immersed in RNAlater, were thawed at room temperature for 0 minute, 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours before RNA extraction. Additionally, tonsil tissue underwent repetitive freezing and thawing cycles. RNA integrity was analyzed by microchip gel electrophoresis and gene expression by quantitative real-time polymerase chain reaction for selected genes (FOS, TGFB1, HIF1A, BCL2, and PCNA). Minimal RNA degradation was detected after 30 minutes of thawing in unfixed samples. This degradation was accompanied by relevant changes in gene expression for FOS and BCL2 at 45 minutes. Modified primer design or the use of different housekeeping genes could not rectify the changes for FOS. Repetitive thawing cycles had similar effects on RNA integrity. The incubation of the tissue in RNAlater efficiently prevented RNA degradation. In conclusion, degradation of RNA in frozen tissue occurs first after several minutes of thawing. Already minimal decrease in RNA quality may result in significant changes in gene expression patterns in clinical tissue samples.

Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens.

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6-16 h. Gene expression by real-time PCR of cfos, HIF1alpha, Bcl2, PCNA, TGFbeta1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2-3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.