Cytotoxic Impact of Naringenin-Loaded Solid Lipid Nanoparticles on RIN5F Pancreatic ß Cells via Autophagy Blockage.

BACKGROUND: Autophagy plays a crucial role in modulating the proliferation of cancer diseases. However, the application of Naringenin (Nar), a compound with potential benefits against these diseases, has been limited due to its poor solubility and bioavailability. OBJECTIVE: This study aimed to develop solid lipid nanoparticles (Nar-SLNs) loaded with Nar to enhance their therapeutic impact. METHODS: In vitro experiments using Rin-5F cells exposed to Nar and Nar-SLNs were carried out to investigate the protective effects of Nar and its nanoformulation against the pancreatic cancer cell line of Rin-5F. RESULTS: Treatment with Nar and Nar-SLN led to an increase in autophagic markers (Akt, LC3, Beclin1, and ATG genes) and a decrease in the level of miR-21. Both Nar and Nar-SLN treatments inhibited cell proliferation and reduced the expression of autophagic markers. Notably, Nar-SLNs exhibited greater efficacy compared to free Nar. CONCLUSION: These findings suggest that SLNs effectively enhance the cytotoxic impact of Nar, making Nar-SLNs a promising candidate for suppressing or preventing Rin-5F cell growth.

Peroxyredoxin 6 Protects RIN-M5F Pancreatic Beta Cells Against Streptozotocin-Induced Senescence.

BACKGROUND/AIMS: There are evidences that a decrease in the functional activity of pancreatic ß-cells under type 2 diabetes conditions may be associated with their senescence, therefore, senotherapy may be a prospective strategy for the diabetes treatment. METHODS: The senotherapeutic potential of peroxiredoxin 6 (PRDX6) was studied in RIN-m5F pancreatic ß-cells with streptozotocin-induced senescence by measuring markers, associated with senescence. RESULTS: Exposure to streptozotocin (STZ) resulted in the senescence of the ß-cells. The addition of PRDX6 to the culture medium of RIN-m5F ß-cells before treatment with STZ decreased the levels of the following senescence markers: the percentage of SA-ß-Gal-positive cells, the phosphorylation of histone H2AX and p21 proteins, and the secretion of the proinflammatory cytokine IL-6 but not the anti-inflammatory cytokine IL-10. These effects were accompanied by a decrease in the production of reactive oxygen species (ROS) and the restoration of impaired NF-κB activation. In addition, PRDX6 altered the production of the heat shock protein HSP90: the production of the constitutive form of HSP90-beta decreased, while the level of inducible HSP90-alpha increased. CONCLUSION: PRDX6 prevented the senescence of RIN-m5F cells in response to the DNA damage-inducing agent streptozotocin, indicating a potential protective role of PRDX6 in type 2 diabetes mellitus.

RIN4 immunity regulators mediate recognition of the core effector RipE1 of Ralstonia solanacearum by the receptor Ptr1.

Ralstonia solanacearum causes lethal bacterial wilt diseases in numerous crops, resulting in considerable yield losses. Harnessing genetic resistance is desirable for safeguarding plants against phytopathogens. However, genetic resources resistant to bacterial wilt are limited in crops. RipE1, a conserved type Ⅲ effector with cysteine protease activity, is recognized in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana). Here, using a virus-induced gene silencing approach, we identified the gene encoding N. benthamiana homologue of Ptr1 (NbPtr1a), a coiled-coil nucleotide-binding leucine-rich repeat receptor (NLR) recognizing RipE1. Silencing or editing NbPtr1a completely abolished RipE1-induced cell death, indicating recognition of RipE1 by NbPtr1a. Genetic complementation confirmed this recognition, which is conserved across multiple solanaceous plants. Expression of RipE1 in planta or within pathogenic bacteria promoted pathogen colonization of Nbptr1a mutant plants, demonstrating its virulence function independent of NLR recognition. Silencing NbRIN4 enhanced RipE1-induced cell death, while expressing NbRIN4 inhibited it, suggesting that NbRIN4 is involved in recognition of NbPtr1a-RipE1. Furthermore, RipE1 associated with and cleaved NbRIN4, AtRIN4, and tomato (Solanum lycopersicum) SlRIN4 proteins through its cysteine protease activity. Silencing NbRIN4 in Nbptr1a mutants did not prevent RipE1 from promoting pathogen colonization, suggesting that NbRIN4 is not the primary target for RipE1-mediated virulence. Additionally, NbRIN4 suppressed self-association of the coiled-coil domain of NbPtr1a, which is critical for NbPtr1a-mediated cell death and resistance. Finally, we demonstrated that activation of NbPtr1a requires RipE1-mediated elimination of NbRIN4. Given the conserved nature of RipE1, Ptr1 holds great potential for protecting crops from diverse R. solanacearum strains and other distinct pathogens.

SlFSR positively regulates ethylene biosynthesis and lycopene accumulation during fruit ripening in tomato.

Transcription factors (TFs) are crucial for regulating fruit ripening in tomato (Solanum lycopersicum). The GRAS (GAI, RGA, and SCR) TFs are involved in various physiological processes, but their role in fruit ripening has seldom been reported. We have previously identified a gene encoding GRAS protein named SlFSR (Fruit Shelf-life Regulator), which is implicated in fruit ripening by regulating cell wall metabolism; however, the underlying mechanism remains unclear. Here, we demonstrate that SlFSR proteins are localized to the nucleus, where they could bind to specific DNA sequences. SlFSR acts downstream of the master ripening regulator RIN and could collaborate with RIN to control the ripening process by regulating expression of ethylene biosynthesis genes. In SlFSR-CR (CRISPR/Cas9) mutants, the initiation of fruit ripening was not affected but the reduced ethylene production and a delayed coloring process occurred. RNA-sequencing (RNA-seq) and promoter analysis reveal that SlFSR directly binds to the promoters of two key ethylene biosynthesis genes (SlACO1 and SlACO3) and activates their expression. However, SlFSR-CR fruits displayed a significant down-regulation of key rate-limiting genes (SlDXS1 and SlGGPPS2) in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which may account for the impaired lycopene synthesis. Altogether, we propose that SlFSR positively regulates ethylene biosynthesis and lycopene accumulation, providing valuable insights into the molecular mechanisms underlying fruit ripening.

Genome assemblies for Chromidotilapia guntheri (Teleostei: Cichlidae) identify a novel candidate gene for vertebrate sex determination, RIN3.

Advances in genome sequencing have greatly accelerated the identification of sex chromosomes in a variety of species. Many of these species have experienced structural rearrangements that reduce recombination between the sex chromosomes, allowing the accumulation of sequence differences over many megabases. Identification of the genes that are responsible for sex determination within these sometimes large regions has proved difficult. Here, we identify an XY sex chromosome system on LG19 in the West African cichlid fish Chromidotilapia guntheri in which the region of differentiation extends over less than 400 kb. We develop high-quality male and female genome assemblies for this species, which confirm the absence of structural variants, and which facilitate the annotation of genes in the region. The peak of differentiation lies within rin3, which has experienced several debilitating mutations on the Y chromosome. We suggest two hypotheses about how these mutations might disrupt endocytosis, leading to Mendelian effects on sexual development.

Crossing a CRISPR/Cas9 transgenic tomato plant with a wild-type plant yields diverse mutations in the F1 progeny.

Generating CRISPR/Cas9-mediated mutants in tomato (Solanum lycopersicum L.) involves screening shoots regenerated from cultured cells transformed with a T-DNA harboring sequences encoding Cas9 and single guide RNAs (sgRNAs). Production of transformants can be inconsistent and obtaining transformants in large numbers may be difficult, resulting in a limited variety of mutations. Here, I report a method for generating various types of mutations from one transgenic plant harboring the CRISPR/Cas9 system. In this method, a wild-type plant was crossed with a T0 biallelic mutant expressing two sgRNAs targeting the RIPENING INHIBITOR (RIN) gene, and the resulting F1 seedlings were classified using a kanamycin resistance marker on the T-DNA. Genotyping of the RIN locus revealed that kanamycin-sensitive F1 seedlings, which carried no T-DNA, always harbored the wild-type allele and a mutant allele from the transgenic parent. Kanamycin-resistant F1 seedlings, which do carry the T-DNA, harbored a variety of novel mutant alleles, but not the wild-type allele, suggesting that it was mutated during crossing. The novel mutations included one-base insertions or short deletions at each target site, or large deletions across the two target sites. This method was also successfully applied to produce mutations in Geranylgeranyl pyrophosphate synthase 2 (GGPS2). Because this method involves crossing rather than transformation, it can be readily scaled up to produce numerous novel mutations, even in plant species or cultivars for which transformation is inefficient. Therefore, when initial transgene experiments fail to induce the desired mutation, this method provides additional opportunities for generating mutants.

Screening of NIAS World Rice Core Collection for Seeds with Long Longevity as Useful Potential Breeding Materials Focusing on the Stability of Embryonic RNAs.

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.

Quality control on oil palm RNA samples for efficient genomic downstream applications.

Quality control (QC) is primordial for determining the efficiency in any downstream genomic applications. There are several steps in the verification of the quality of RNA samples destined for genomic studies. The aim of this research was to determine whether RNA should be discarded at the level of the field lab if it fails preliminary quality control using Optical Density (OD) measurements. In this study, all samples were submitted to rigorous quality control in every stage of work. RNA samples showing poor OD values still gave excellent results in downstream QC and genomic applications. At the end of the quality control exercise, it was observed that the original samples were the same and had not undergone any deterioration along the different stages of handling and manipulation. This paper shows the different and most important stages of quality control on RNA samples (RIN) for an effective down stream application in genomic studies. RNA samples should not be discarded based on preliminary QC from our field labs.

Defective mutations in STAY-GREEN 1, PHYTOENE SYNTHASE 1, and MYB12 genes lead to formation of green ripe fruit in tomato.

Modern tomatoes produce colorful mature fruits, but many wild tomato ancestors form green or gray green ripe fruits. Here, tomato cultivar 'Lvbaoshi' (LBS) that produces green ripe fruits was found to contain three recessive loci responsible for fruit development. The colorless peel of LBS fruits was caused by a 603 bp deletion in the promoter of SlMYB12. The candidate genes of the remaining two loci were identified as STAY-GREEN 1 (SlSGR1) and PHYTOENE SYNTHASE 1 (SlPSY1). SGR1 and PSY1 co-suppression by RNAi converted the pink fruits into green ripe fruits in transgenic plants. An amino acid change in PSY1 and a deletion in the promoter of SGR1 were also identified in several wild tomatoes bearing green or gray ripe fruits. Overexpression of PSY1 from green ripe fruit wild tomatoes in LBS plants could only partially rescue the green ripe fruit phenotype of LBS, and transgenic lines expressing ProSGR1::SGR1 from Solanum pennellii also failed to convert purple-flesh into red-flesh fruits. This work uncovers a novel regulatory mechanism by which SlMYB12, SlPSY1, and SlSGR1 control fruit color in cultivated and some wild tomato species.

The RIN4-like/NOI proteins NOI10 and NOI11 modulate the response to biotic stresses mediated by RIN4 in Arabidopsis.

KEY MESSAGE: NOI10 and NOI11 are two RIN4-like/NOI proteins that participate in the immune response of the Arabidopsis plant and affect the RIN4-regulated mechanisms involving the R-proteins RPM1 and RPS2. The immune response in plants depends on the regulation of signaling pathways triggered by pathogens and herbivores. RIN4, a protein of the RIN4-like/NOI family, is considered to be a central immune signal in the interactions of plants and pathogens. In Arabidopsis thaliana, four of the 15 members of the RIN4-like/NOI family (NOI3, NOI5, NOI10, and NOI11) were induced in response to the plant herbivore Tetranychus urticae. While overexpressing NOI10 and NOI11 plants did not affect mite performance, opposite callose accumulation patterns were observed when compared to RIN4 overexpressing plants. In vitro and in vivo analyses demonstrated the interaction of NOI10 and NOI11 with the RIN4 interactors RPM1, RPS2, and RIPK, suggesting a role in the context of the RIN4-regulated immune response. Transient expression experiments in Nicotiana benthamiana evidenced that NOI10 and NOI11 differed from RIN4 in their functionality. Furthermore, overexpressing NOI10 and NOI11 plants had significant differences in susceptibility with WT and overexpressing RIN4 plants when challenged with Pseudomonas syringae bacteria expressing the AvrRpt2 or the AvrRpm1 effectors. These results demonstrate the participation of NOI10 and NOI11 in the RIN4-mediated pathway. Whereas RIN4 is considered a guardee protein, NOI10 and NOI11 could act as decoys to modulate the concerted activity of effectors and R-proteins.

Microarray Analysis to Determine Gene Expression Changes in Zebrafish Embryos.

Chemical exposure in humans begins from the zygote stage and continues throughout the development of the embryo and the fetus. Zebrafish are one of the most powerful model organisms used in many research areas, including genetics, environmental toxicology, development, DNA damage and repair, cancer, and other diseases. Among the advantages that facilitate the use of zebrafish as a model for studies are features such as high homology with the human genome, small size, and high reproductive potential in short periods. The use of zebrafish embryos in research has increased rapidly due to their advantageous properties, including extrauterine development and the transparent feature of the embryos. However, there are thousands of genes that can be encountered in research, and in this case, the workforce is too much. This workload has been alleviated with the developed technologies. Microarray is one of these technologies. An important parameter in this assay is the RIN value. The RIN value ranges from 1 to 10, indicating mRNA degradation, and therefore helps to decide whether to continue the study. In this chapter, microarray analysis, which is one of the main techniques used in the determination of gene expression in zebrafish embryos, is described.

Exploring the potential of Lindernia ciliata: isolation, characterization, and pharmacological evaluation of its anti-diabetic properties.

We isolated two phytoconstituents using bioassay guided isolation for anti-diabetic property from the hydroalcoholic extract of Lindernia ciliata (Colsm.) Pennell (Family: Linderniaceae). We assessed the anti-diabetic potential using various assays, including the glucose absorption assay, glucose uptake in isolated rat abdominal muscle assay, insulin secretion by RIN-5F cells assay, α-amylase inhibition activity, and DPP-4 inhibition assay. The results from our study indicated that the ethanol and water fractions obtained from the hydroalcoholic extract significantly improved glucose uptake, demonstrating promising anti-diabetic properties. Further investigation led to the isolation of two distinct compounds, LCF-1 (IUPAC name: 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) (LCF-1) and LCF-2 (IUPAC name: 3S,10R,13R,17R)-17-[(2R,5R)-5-ethyl-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol). Consequently, our findings suggest that LCF-1 and LCF-2 are two phytoconstituent derived from the hydroalcoholic extract of L. ciliata, could serve as a potential source of anti-diabetic agents. This highlights its suitability for further development in the creation of herbal pharmaceuticals for diabetes treatment.

Introgression of a dominant phototropin1 mutant enhances carotenoids and boosts flavour-related volatiles in genome-edited tomato RIN mutants.

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation is known to completely repress fruit ripening. The heterozygous (RIN/rin) fruits have extended shelf life, ripen normally, but have inferior taste/flavour. To address this, we used genome editing to generate newer alleles of RIN (rinCR ) by targeting the K-domain. Unlike previously reported CRISPR alleles, the rinCR alleles displayed delayed onset of ripening, suggesting that the mutated K-domain represses the onset of ripening. The rinCR fruits had extended shelf life and accumulated carotenoids at an intermediate level between rin and progenitor line. Besides, the metabolites and hormonal levels in rinCR fruits were more akin to rin. To overcome the negative attributes of rin, we crossed the rinCR alleles with Nps1, a dominant-negative phototropin1 mutant, which enhances carotenoid levels in tomato fruits. The resulting Nps1/rinCR hybrids had extended shelf life and 4.4-7.1-fold higher carotenoid levels than the wild-type parent. The metabolome of Nps1/rinCR fruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatiles in Nps1/rinCR were only observed in fruits bearing the homozygous Nps1 mutation. The Nps1 introgression into tomato provides a promising strategy for developing cultivars with extended shelf life, improved taste, and flavour.

The Role of RIN3 Gene in Alzheimer's Disease Pathogenesis: a Comprehensive Review.

Alzheimer's disease (AD) is a globally prevalent form of dementia that impacts diverse populations and is characterized by progressive neurodegeneration and impairments in executive memory. Although the exact mechanisms underlying AD pathogenesis remain unclear, it is commonly accepted that the aggregation of misfolded proteins, such as amyloid plaques and neurofibrillary tau tangles, plays a critical role. Additionally, AD is a multifactorial condition influenced by various genetic factors and can manifest as either early-onset AD (EOAD) or late-onset AD (LOAD), each associated with specific gene variants. One gene of particular interest in both EOAD and LOAD is RIN3, a guanine nucleotide exchange factor. This gene plays a multifaceted role in AD pathogenesis. Firstly, upregulation of RIN3 can result in endosomal enlargement and dysfunction, thereby facilitating the accumulation of beta-amyloid (Aß) peptides in the brain. Secondly, RIN3 has been shown to impact the PICLAM pathway, affecting transcytosis across the blood-brain barrier. Lastly, RIN3 has implications for immune-mediated responses, notably through its influence on the PTK2B gene. This review aims to provide a concise overview of AD and delve into the role of the RIN3 gene in its pathogenesis.

P2Y12 receptor residues crucial for thrombosis regulation.

Thrombosis, or the formation of blood clots, can lead to serious medical conditions such as stroke, heart attack, and deep vein thrombosis. The purinoreceptor P2Y12 plays a critical role in the thrombotic pathway and is targeted for therapy to prevent clot formation. However, it is essential to balance the regulation of thrombosis to avoid adverse situations. This study focuses on the P2Y12 receptor and aims to discern the protein residue network and differentiate residues based on their intramolecular interactions. The study utilized a statistical analysis to characterize the significant residues involved in ligand interaction, which helps to identify critical residues that are essential for the function of the receptor. A parametric analysis of interactions of residues in the intraprotein interaction was conducted, which revealed significant residue-based contacts that facilitate protein interactions. By examining the interactions between residues, the mechanisms underlying protein interactions were studied and the importance of specific residues in facilitating these interactions was determined. This research provides important information on P2Y12, and the findings based on the network based significance of interacting residues may contribute to the development of new therapies that target the receptor to prevent clot formation while maintaining a balance in thrombosis regulation to avoid adverse outcomes. Ultimately, this study could lead to improved treatments for thrombotic disorders and better patient outcomes.

Rinmaker: a fast, versatile and reliable tool to determine residue interaction networks in proteins.

BACKGROUND: Residue Interaction Networks (RINs) map the crystallographic description of a protein into a graph, where amino acids are represented as nodes and non-covalent bonds as edges. Determination and visualization of a protein as a RIN provides insights on the topological properties (and hence their related biological functions) of large proteins without dealing with the full complexity of the three-dimensional description, and hence it represents an invaluable tool of modern bioinformatics. RESULTS: We present RINmaker, a fast, flexible, and powerful tool for determining and visualizing RINs that include all standard non-covalent interactions. RINmaker is offered as a cross-platform and open source software that can be used either as a command-line tool or through a web application or a web API service. We benchmark its efficiency against the main alternatives and provide explicit tests to show its performance and its correctness. CONCLUSIONS: RINmaker is designed to be fully customizable, from a simple and handy support for experimental research to a sophisticated computational tool that can be embedded into a large computational pipeline. Hence, it paves the way to bridge the gap between data-driven/machine learning approaches and numerical simulations of simple, physically motivated, models.

Lycium RIN negatively modulate the biosynthesis of kukoamine A in hairy roots through decreasing thermospermine synthase expression.

Root bark (Lycii cortex) of Lycium contains high contents of characteristic bioactive compounds, including kukoamine A (KuA) and kukoamine B (KuB). RIPENING INHIBITOR (RIN) is well known as a master regulator of Solanaceaous fruit ripening. However, the role of RIN in the biosynthetic pathway of KuA in Lycium remains unclear. In this study, integrated transcriptomic, metabolomic analyses and hairy root system are used to characterize the role of RIN in KuA biosynthesis in Lycium. The ultra performance liquid chromatography electrospray ionization tandem mass spectrometry analysis revealed that KuA was significantly induced in LrRIN1 RNAi lines and not detected in overexpression lines. A total of 20,913 differentially expressed genes (DEGs) and 60 differentially accumulated metabolites (DAMs) were detected in LrRIN1 transgenic hairy roots, which were used for weighted gene co-expression network analysis. Our result reveals a high association between KuA and structural genes in the phenolamide pathway, which shows a negative correlation with LrRIN1. In addition, overexpression of the polyamine pathway gene thermospermine synthase LcTSPMS, a potential target gene of Lycium RIN, increased the contents of both KuA and KuB in L. chinense hairy root, indicating that TSPMS is responsible for KuA biosynthesis and is also the common upstream biosynthetic gene for both KuA and KuB. Our results lay a solid foundation for uncovering the biosynthetic pathway of KuA, which will facilitate the molecular breeding and genetic improvement of Lycium species.

Swiss Vascular Biobank: Evaluation of Optimal Extraction Method and Admission Solution for Preserving RNA from Human Vascular Tissue.

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.

High purity and integrity RNA from human cell lines stored in liquid nitrogen for over 20 years.

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.