Seed longevity is a crucial trait for the seed industry and genetic resource preservation. To develop excellent cultivars with extended seed lifespans, it is important to understand the mechanism of keeping seed germinability long term and to find useful genetic resources as prospective breeding materials. This study was conducted to identify the best cultivars with a high and stable seed longevity trait in the germplasm of rice (Oryza sativa L.) and to analyze the correlation between seed longevity and embryonic RNA integrity. Seeds from 69 cultivars of the world rice core collection selected by the NIAS in Japan were harvested in different years and subjected to long-term storage or controlled deterioration treatment (CDT). The long-term storage (4 °C, RH under 35%, 10 years) was performed on seeds harvested in 2010 and 2013. The seeds harvested in 2016 and 2019 were used for CDT (36 °C, RH of 80%, 40 days). Seed longevity and embryonic RNA integrity were estimated by a decrease in the germination percentage and RNA integrity number (RIN) after long-term storage or CDT. The RIN value was obtained by the electrophoresis of the total RNA extracted from the seed embryos. Seeds of \"Vandaran (indica)\", \"Tupa 729 (japonica)\", and \"Badari Dhan (indica)\" consistently showed higher seed longevity and embryonic RNA integrity both under long-term storage and CDT conditions regardless of the harvest year. A strong correlation (R2 = 0.93) was observed between the germination percentages and RIN values of the seeds after the long-term storage or CDT among nine cultivars selected based on differences in their seed longevity. The study findings revealed the relationship between rice seed longevity and embryo RNA stability and suggested potential breeding materials including both japonica and indica cultivars for improving rice seed longevity.

Proper biobanking is essential for obtaining reliable data, particularly for next-generation sequencing approaches. Diseased vascular tissues, having extended atherosclerotic pathologies, represent a particular challenge due to low RNA quality. In order to address this issue, we isolated RNA from vascular samples collected in our Swiss Vascular Biobank (SVB); these included abdominal aortic aneurysm (AAA), peripheral arterial disease (PAD), healthy aorta (HA), and muscle samples. We used different methods, investigated various admission solutions, determined RNA integrity numbers (RINs), and performed expression analyses of housekeeping genes (ACTB, GAPDH), ribosomal genes (18S, 28S), and long non-coding RNAs (MALAT1, H19). Our results show that RINs from diseased vascular tissue are low (2-4). If the isolation of primary cells is intended, as in our SVB, a cryoprotective solution is a better option for tissue preservation than RNAlater. Because RNA degradation proceeds randomly, controls with similar RINs are recommended. Otherwise, the data might convey differences in RNA degradation rather than the expressions of the corresponding genes. Moreover, since the 18S and 28S genes in the diseased vascular samples were degraded and corresponded with the low RINs, we believe that DV200, which represents the total RNA\'s disintegration state, is a better decision-making aid in choosing samples for omics analyses.

Biobanks are a key resource for obtaining human cell lines for biomedical research, including for drug development projects. Such projects often include comparative RNA-sequencing of large panels of human cell lines from individuals affected by certain disorders and healthy controls, or from individuals with different drug response phenotypes. RNA extractions are typically done from growing cell cultures, a process that may take several weeks. However, maintaining large numbers of cell lines in parallel increases the project workload. Here, we show that extracting RNAs directly from frozen vials of human cell lines stored for over 20 years in a liquid nitrogen freezer yields RNAs with the high purity and integrity parameters that conform to those required for optimal RNA-sequencing and are closely similar to those obtained for RNAs extracted from growing human cell lines.

Background: RNA extracted from human blood has been widely applied to biological, medical, and clinical research of numerous diseases. Previous studies have demonstrated that high-quality RNA is indispensable to guarantee the reliability of downstream assays. In this study, we investigated the effects of freezing procedures, rewarming methods, and blood components on RNA quality of blood samples. Methods: Rabbit blood samples were divided into two groups: (1) whole blood (WB) and (2) blood cell components (BCC) with plasma removed. Samples were frozen using four representative freezing procedures (snap freezing in liquid nitrogen, snap freezing at -80°C, traditional slow freezing, and programmable controlled rate freezing) and rewarmed by placing at 4°C or by vortexing. RNA was extracted using the phenol-chloroform RNA extraction method and measured by an Agilent bioanalyzer. Then, human blood was used to verify the best protocol obtained from the rabbit blood experiment. Results: For the four freezing procedures, there were no differences in RNA integrity. For different rewarming methods, RNA integrity number (RIN) values of RNA extracted from frozen WB and BCC samples in the vortex group were above 9, while RNA obtained from WB showed worse quality compared with BCC in the 4°C group. For verification using human blood, RIN values of frozen human WB rewarmed by vortexing ranged from 8.0 to 9.1. Conclusions: Blood components and rewarming methods could affect the RNA quality of blood samples. For scenarios where WB samples have already been cryopreserved, the vortex rewarming method is optimal for high-quality RNA. Otherwise, we would recommend centrifuging fresh WB and cryopreserving it in the form of BCC, which showed a tendency to obtain high-quality RNA by either of the two rewarming methods.

Cryopreserving tissues for histology requires the use of coolants to buffer the sample from liquid nitrogen (LN2) and to control the rate of temperature decline. Several coolants sharing similar physical characteristics are available on the market; however, commonly used coolants are variably flammable and/or toxic and pose risks to personnel and facilities. The purpose of this study was to compare the performance of three commercially available coolants: hexane, 2-methylbutane (2 M), and 1-methoxyheptafluoropropane (N7000). Fresh mouse tissues were frozen by each method, for their ability to preserve microscopic architecture and to protect RNA from degradation were evaluated and compared to tissue characteristics obtained by direct immersion in LN2. Our results show that for most tissues, the N7000 freezing coolant provides equal or improved preservation of microscopic architecture. While snap-freezing tissues in LN2 provides superior RNA protection, no significant differences in RNA quality were seen between tissues frozen in hexane, 2 M, and N7000.

The mature embryos of rice seeds contain translatable mRNAs required for the initial phase of germination. To clarify the relationship between seed longevity and RNA integrity in embryos, germinability and stability of embryonic RNAs were analyzed using the seeds of japonica rice cultivars subjected to controlled deterioration treatment (CDT) or long periods of storage. Degradation of RNA from embryos of a japonica rice cultivar \"Nipponbare\" was induced by CDT before the decline of the germination rate and we observed a positive relationship between seed germinability and integrity of embryonic RNAs. Moreover, this relationship was confirmed in the experiments using aged seeds from the \"Nipponbare\", \"Sasanishiki\" and \"Koshihikari\" rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R2=0.75). Therefore, the stability of embryonic RNAs required for germination is involved in maintaining seed longevity over time and RIN values can serve as a quantitative indicator to evaluate germinability in rice.

Background: High-quality RNA extraction from tissue samples is of key importance for scientific research and translational medicine. Tissue collection and preparation may affect RNA quality. In this study, we investigated effects of warm ischemia time, cryopreservation, and grinding methods on RNA quality. Methods: Total RNA was extracted from mouse kidney tissues with warm ischemia times of 0, 30, 60, 90, and 120 minutes. Half of the tissues were used to extract RNA immediately, while the others were cryopreserved in the vapor phase of liquid nitrogen for 6 months before RNA extraction. A mortar, homogenizer, and tissue lyser were used to grind tissues. RNA was extracted by TRIzol, and RNA integrity was assessed by the RNA integrity number (RIN) value. Results: For fresh tissues and frozen tissues with warm ischemia time within 60 minutes, RIN values were above 7.0 and remained above 6.0 with warm ischemia time within 120 minutes. For the same warm ischemia time, RIN values of frozen tissues were slightly lower than those of fresh tissues. No significant RIN value alterations were observed among grinding methods, but for RNA extraction efficiency, a mortar was much less efficient than the homogenizer or tissue lyser. For frozen tissues, RNA tended to degrade within 8 minutes at room temperature. Conclusions: Mouse kidney tissues with a warm ischemia time within 120 minutes are suitable for general RNA-related research. For tissues with a warm ischemia time within 60 minutes, cryopreservation may not affect RNA quality. The duration of frozen tissues held at room temperature before grinding affects the integrity of RNA, while grinding methods do not affect RNA integrity.

Gene expression studies are reported to be influenced by pre-analytical factors that can compromise RNA yield and integrity, which in turn may confound the experimental findings. Here we investigate the impact of four pre-analytical factors on brain-derived RNA: time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method. We report no significant differences in RNA yield or integrity between 20 mg and 60 mg tissue samples collected in either liquid nitrogen or the RNAlater stabilizing solution. Isolation of RNA employing the TRIzol reagent resulted in a higher yield compared to isolation via the QIAcube kit while the latter resulted in RNA of slightly better integrity. Keeping brain tissue samples at room temperature for up to 160 min prior to collection and isolation of RNA resulted in no significant difference in yield or integrity. Our findings have significant practical and financial consequences for clinical genomic departments and other laboratory settings performing large-scale routine RNA expression analysis of brain samples.

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

A suitable technique (lysozyme combined with Trizol reagent) was developed to improve the RNA yield, purity and integrity from Bacillus subtilis, under different bacterial growth stages. The obtained RNA was intact, having the required characteristics for downstream applications.