Integrative Analysis of Genome, 3D Genome, and Transcriptome Alterations of Clinical Lung Cancer Samples.

Genomic studies of cancer cell alterations, such as mutations, copy number variations (CNVs), and translocations, greatly promote our understanding of the genesis and development of cancers. However, the 3D genome architecture of cancers remains less studied due to the complexity of cancer genomes and technical difficulties. To explore the 3D genome structure in clinical lung cancer, we performed Hi-C experiments using paired normal and tumor cells harvested from patients with lung cancer, combining with RNA sequenceing analysis. We demonstrated the feasibility of studying 3D genome of clinical lung cancer samples with a small number of cells (1 × 104), compared the genome architecture between clinical samples and cell lines of lung cancer, and identified conserved and changed spatial chromatin structures between normal and cancer samples. We also showed that Hi-C data can be used to infer CNVs and point mutations in cancer. By integrating those different types of cancer alterations, we showed significant associations between CNVs, 3D genome, and gene expression. We propose that 3D genome mediates the effects of cancer genomic alterations on gene expression through altering regulatory chromatin structures. Our study highlights the importance of analyzing 3D genomes of clinical cancer samples in addition to cancer cell lines and provides an integrative genomic analysis pipeline for future larger-scale studies in lung cancer and other cancers.

Obtaining High-Quality Blood Specimens for Downstream Applications: A Review of Current Knowledge and Best Practices.

Blood is a biological fluid that contains multiple blood fraction and cellular components. High-quality blood specimens are essential prerequisites for various downstream applications such as molecular epidemiology studies, genomics, and proteomics studies. Currently, protocols and research publications concerning the collection, handling, preservation, and stability of blood or blood fractions are constantly emerging. Moreover, standardized guidelines are a requirement for biorepositories to tightly control preanalytical variables originating from these procedures and obtain high-quality blood specimen for downstream analyses. In this review article, we summarize the best practices and fit-for-purpose protocols regarding blood collection, processing, storage, and stability. In addition, we present some typical quality biomarkers, which could be used to evaluate the integrity of blood specimens.

Long-Term Room Temperature Storage of Dry Ribonucleic Acid for Use in RNA-Seq Analysis.

RNA is an essential biological material for research in genomics and translational medicine. As such, its storage for biobanking is an important field of study. Traditionally, long-term storage in the cold (generally freezers or liquid nitrogen) is used to maintain high-quality (in terms of quantity and integrity) RNA. Room temperature (RT) preservation provides an alternative to the cold, which is plagued by serious problems (mainly cost and safety), for RNA long-term storage. In this study, we evaluated the performance of several RT storage procedures, including the RNAshell® from Imagene, where the RNA is dried and kept protected from the atmosphere, and the vacuum drying of RNA with additives such as the Imagene stabilization solution and a home-made trehalose solution. This evaluation was performed through accelerated (equivalent to 10 years for RNAshell) aging and real-time studies (4 years). To check RNA quality and integrity, we used RNA integrity number values and RNA-seq. Our study shows that isolation from atmosphere offers a superior protective effect for RNA storage compared with vacuum drying alone, and demonstrates that RNAshell permits satisfactory RNA quality for long-term RT storage. Thus, the RNA quality could meet the demand of downstream applications such as RNA-seq.

Evaluation of DNA/RNAshells for room temperature nucleic acids storage.

Traditional nucleic acids preservation methods rely on maintaining samples in cold environments, which are costly to operate and time sensitive. Recent work validated that using room temperature for the storage of nucleic acids is possible if the samples are completely protected from water and oxygen. Here, we conducted accelerated aging and real-time degradation studies to evaluate the new technology DNAshell and RNAshell, which preserves DNA and RNA at room temperature, including the DNA and RNA yield, purity, and integrity. DNA and RNA solutions are dried in the presence of stabilizers in stainless steel minicapsules, then redissolved after different time points of heating and storing at room temperature. Results show that DNAshell and RNAshell ensure the safe storage of nucleic acids at room temperature for long periods of time, and that the quality of these nucleic acids is suitable for common downstream analysis.

Comparison of six different pretreatment methods for blood RNA extraction.

Human blood specimens serve as important research materials in the field of translational medicine research. The RNA extracted from blood, for example, represents the gene expression profiles of individuals or groups, and can be indicative of the pathological basis for human diseases. Meanwhile, the RNA quality may have severe impacts on the results of RNA studies. RNA is susceptible to many factors, such as the time of sample collection, transportation conditions, protectants, pretreatments, and extraction methods. In this study, six different pretreatment methods are evaluated for their effects on blood RNA extraction including the RNA yields and quality. Results show that most of these methods meet the basic requirements for RNA studies. While considering the simplicity of the procedure, the cost factor, and how to make full use of the samples, the proper method should be employed by researchers who have specific requirements for their research.