Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at ?80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored.     

RNA Stability in Human Liver: Comparison of Different Processing Times, Temperatures and Methods

The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1?? and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank.     

Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis

Fine-needle aspirate samples hold the potential for gaining valuable insight into the molecular details and prognostic indicators for certain types of cancer in a limited volume of relatively pure tumor cells. Although limited, such clinical samples can be used with high efficiency when analyzed in conjunction with gene-dense expression microarrays. For this reason, it is essential to retrieve as much high-quality genetic material as possible from each fine-needle aspirate sample. We have conducted a study to improve the efficiency of extracting high quality total RNA to use in microarray analysis from single ex vivo fine-needle aspirate samples of 11 breast cancers added to RNAlater RNA Stabilization Reagent immediately upon collection. Approximately half the total RNA from fine-needle aspirate samples of breast cancers was isolated from the supernatant, and that RNA had similar quality and gene expression profile to the RNA that was isolated from the corresponding cell pellet. We recommend that the supernatant not be discarded when extracting RNA from fine-needle aspirate samples stored in RNAlater.     

High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues

Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater, which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality afterflow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissues.     

Proteomics reveals different pathological processes of adipose tissue,liver, and skeletal muscle under insulin resistance

Type 2 diabetes mellitus is the most common type of diabetes, and insulin resistance (IR) is its core pathological mechanism. Proteomics is an ingenious and promising Omics technology that can comprehensively describe the global protein expression profiling of body or specific tissue, and is widely applied to the study of molecular mechanisms of diseases. In this paper, we focused on insulin target organs: adipose tissue, liver, and skeletal muscle, and analyzed the different pathological processes of IR in these three tissues based on proteomics research. By literature studies, we proposed that the main pathological processes of IR among target organs were diverse, which showed unique characteristics and focuses. We further summarized the differential proteins in target organs which were verified to be related to IR, and discussed the proteins that may play key roles in the emphasized pathological processes, aiming at discovering potentially specific differential proteins of IR, and providing new ideas for pathological mechanism research of IR.