Amount Of Total RNA Research Articles

RNA Extraction from Rice Immature Embryo Using Laser Capture Microdissection.

Laser capture microdissection (LCM) enables the selective isolation of organs, tissues, and cells from surrounding tissues. Total RNA extracted from small tissue sections can be used for a variety of subsequent analysis such as RNA-seq analysis. Here, we describe a method for isolating embryos from rice ovary sections using LCM and extracting total RNA. We successfully obtained sufficient amount of total RNA from a single rice embryo at the globular embryo stage for RNA-seq analysis.

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P > 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparative Analysis of Viromes Identified in Multiple Macrofungi.

Macrofungi play important roles in the soil elemental cycle of terrestrial ecosystems. Fungal viruses are common in filamentous fungi, and some of them can affect the growth and development of hosts. However, the composition and evolution of macrofungal viruses are understudied. In this study, ninety strains of Trametes versicolor, Coprinellus micaceus, Amanita strobiliformis, and Trametes hirsuta were collected in China. Four mixed pools were generated by combining equal quantities of total RNA from each strain, according to the fungal species, and then subjected to RNA sequencing. The sequences were assembled, annotated, and then used for phylogenetic analysis. Twenty novel viruses or viral fragments were characterized from the four species of macrofungi. Based on the phylogenetic analysis, most of the viral contigs were classified into ten viral families or orders: Barnaviridae, Benyviridae, Botourmiaviridae, Deltaflexiviridae, Fusariviridae, Hypoviridae, Totiviridae, Mitoviridae, Mymonaviridae, and Bunyavirales. Of these, ambi-like viruses with circular genomes were widely distributed among the studied species. Furthermore, the number and overall abundance of viruses in these four species of macrofungi (Basidiomycota) were found to be much lower than those in broad-host phytopathogenic fungi (Ascomycota: Sclerotinia sclerotiorum, and Botrytis cinerea). By employing metatranscriptomic analysis in this study, for the first time, we demonstrated the presence of multiple mycoviruses in Amanita strobiliformis, Coprinellus micaceus, Trametes hirsute, and Trametes versicolor, significantly contributing to research on mycoviruses in macrofungi.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

Comparative RNA isolation methods from fresh ejaculated spermatozoa in Sahiwal cattle (Bos indicus) and Murrah buffalo (Bubalus bubalis) bulls for high quality and enhanced RNA yield

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. The aim of the study was to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bulls. Ten fresh ejaculates from each Sahiwal (n = 10 bulls × 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) resulted significantly (p < .05) higher total RNA quantity (300–340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30–40 million, 70–80 million and 300–400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm.

RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.

Data-driven identification of total RNA expression genes for estimation of RNA abundance in heterogeneous cell types highlighted in brain tissue

We define and identify a new class of control genes for next-generation sequencing called total RNA expression genes (TREGs), which correlate with total RNA abundance in cell types of different sizes and transcriptional activity. We provide a data-driven method to identify TREGs from single-cell RNA sequencing data, allowing the estimation of total amount of RNA when restricted to quantifying a limited number of genes. We demonstrate our method in postmortem human brain using multiplex single-molecule fluorescent in situ hybridization and compare candidate TREGs against classic housekeeping genes. We identify AKT3 as a top TREG across five brain regions.

Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes†.

Numerous reference genes for use with quantitative reverse transcription polymerase chain reaction (RT-qPCR) have been used for oocytes, eggs, and preimplantation embryos. However, none are actually suitable because of their large variations in expression between developmental stages. To address this, we produced a standardized and merged RNA sequencing (RNAseq) data set by combining multiple publicly available RNAseq data sets that spanned mouse GV oocytes, MII eggs, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos to identify transcripts with essentially constant expression across all stages. Their expression was then measured using RT-qPCR, with which they did not exhibit constant expression but instead revealed a fixed quantitative relationship between measurements by the two techniques. From this, the relative amounts of total messenger RNA at each stage from the GV oocyte through blastocyst stages were calculated. The quantitative relationship between measurements by RNAseq and RT-qPCR was then used to find genes predicted to have constant expression across stages in RT-qPCR. Candidates were assessed by RT-qPCR to confirm constant expression, identifying Hmgb3 and Rb1cc1 or the geometric mean of those plus either Taf1d or Cd320 as suitable reference genes. This work not only identified transcripts with constant expression from mouse GV oocytes to blastocysts, but also determined a general quantitative relationship between expression measured by RNAseq and RT-qPCR across stages that revealed the relative levels of total mRNA at each stage. The standardized and merged RNA data set should also prove useful in determining transcript expression in mouse oocytes, eggs, and embryos.

Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa.

Being highly fragmented and low in concentration, isolation of good quality RNA from sperm cells is a big challenge. Attempts have been made to evaluate various sperm RNA isolation methods from purified buffalo bull sperm cells. Both, non-membrane and membrane-based methods have been evaluated for isolating RNA from Murrah buffalo sperms and compared for their respective efficacies. The traditional TRIzol, TRIzol-heat lysed (H-TRIzol) and cocktail of TCEP-RLT lysis buffer (Qiagen RNeasy mini kit)-TRIzol (C-TRIzol) based isopropanol isolation methods have been evaluated. H-TRIzol yielded best results among conventional methods. The combined T-RLT RNA isolation protocol yielded best quality and quantity compared to other membrane-based methods, due to high lytic property of cocktail of lysis reagents, necessary for complete breakdown of sperm membrane and RNA binding membrane for RNA isolation. Combined lysis performed by treatment with RLT-T and T-RLT differing in order of reagents used were also evaluated. T-RLT combination giving better results compared to RLT-T due to high gDNA contamination and membrane clogging in later protocol steps. Overall, in terms of total RNA quantity and quality per million spermatozoa, the heat-lysed TRIzol method (H-TRIzol) performs best among RNA separation techniques employed and is also quite easy to perform. This comparative evaluation of sperm RNA isolation protocols can be useful in deciding the best protocol for isolation of good quality and high concentration sperm RNA from buffalo semen, for transcriptome and other downstream studies.

Exploring the Possibility of RNA in Diverse Biological Processes

The total amount of RNA in a cell is 5 to 10 times greater than that of DNA [...]

Site-specific analysis of ribosomal 2'O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations.

Ribose 2'O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2'O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.

Gene Expression Analyses in Human Follicles.

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff's laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 °C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 °C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 µm) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/µL. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China Zhenggang Li, Yafei Tang, Xiaoman She, Guobing Lan, Lin Yu, and Zifu He† Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P. R. China. Tomato yellow mottle-associated virus (TYMaV) is a newly found cytorhabdovirus associated with epinasty of leaflet blades, yellow spots, puckering, and mottling symptoms in tomato plants in China (Xu et al., 2017). In May 2020, Solanum nigrum plants exhibiting leaf crinkling and mosaic symptoms (eXtra S1) were found in Shantou city, Guangdong, China. To identify the causal pathogens, the leaves of three symptomatic plants were collected and subjected to total RNA extraction with TRIzol Reagent (Takara, Kusatsu, Japan). About 100 μg RNA mixture, which consisted of an equal amount of total RNA extracted from the three samples, was subjected to small RNA deep sequencing and assembly (sRSA) (Kreuze et al., 2009). Small RNA cDNA library was constructed with the method described previously (Mi et al., 2008). Small RNA deep sequencing was performed with Illumina HiSeq X Ten platform. VirusDetect (Zheng et al., 2017) was used to analyze the sequence data. The result showed that the sequence data includes about 11 million reads and generated 194 unique contigs after removal of host-derived contigs. Subsequently, the unique contigs were screened using BLASTn search against the virus database. One hundred and five unique contigs were mapped to TYMaV genome (reference sequence, KY075646), 21 unique contigs were mapped to RNA1 segment of tomato chlorosis virus (ToCV) genome (reference sequence, KY618796), 67 unique contigs were mapped to RNA2 segment of ToCV genome (reference sequence, KY618797), and one unique contig was mapped to pepper veinal mottle virus (PVMV) genome (reference sequence, FJ617225) (eXtra S1). To verify the sRSA result for TYMaV detection, RT-PCR was performed with two primer pairs TYMaV-F1/R1 (5'-TCATTAGACTCAGGCCTAATCCTCA AAGT-3'/5'-GATATGGAGACGTCCAAGTTCAAAGGGATGGA-3'), and TYMaV-F2/R2 (5'-TATGCGGCAGCTTTCATGTCTATAGACCCT-3'/5'-ATGACCTAGCTTCAATAACAGTCGCG-3'), which are designed according to the sRSA result. All the symptomatic samples tested positive for TYMaV (eXtra S2). Western blot with TYMaV N protein-specific antibody further verified the result (eXtra S2). To obtain the nearly full-length sequence of TYMaV identified in Shantou, 13 primer pairs were designed to amplify the viral fragments. The amplified PCR products were then introduced into pMD19T (Takara, Kusatsu, Japan) and sequenced by Sangon Biotech Co. (Shanghai, China). The nearly full-length sequence of TYMaV Shantou isolate (TYMaV-ST) was assembled from the 13 overlapping sequences (reference sequence, MW527091). TYMaV-ST genome comprises of 13401 nt and shares 84.93% nucleotide sequence identity with the reference genome (KY075646). In addition, 37 S. nigrum samples and 20 tomato samples nearby with viral disease symptoms were collected from different sites of Guangdong province, China. Six S. nigrum samples and five tomato plant samples tested positive for TYMaV by RT-PCR, suggesting a wide spread of the virus in the surveyed region. These results together with those of the sRSA assay also suggest that the disease symptoms shown in the original S. nigrum plants may not necessarily be caused by TYMaV or by TYMaV alone. To our knowledge, this is the first report of TYMaV infecting S. nigrum in China. S. nigrum is a common weed which belongs to the family Solanaceae and may serve as a reservoir for TYMaV in the fields. Further research is needed to verify whether this is indeed the case, and to understand the characteristics of this virus including its transmission, pathogenicity, and economic significance. The authors declare no conflict of interest. Funding This work was supported by the Key Research and Development Program of Guangdong Province (2018B020202006), the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences (202103TD and 202105TD), the Science and Technology Program of Guangzhou (202102020504), and Special Fund for Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science (R2019PY-QF003).

Effects on expression of B lymphocyte transmembrane ligand genes after treatment with Rose extract

The goal was to evaluate the role of red rose extract (Pierre de Ronsard) on the human B lymphocytes gene CD20, CD30, CD40, and CCR5 expression. Red rose extract was prepared at the dilution of 0.0075% (v/v) and stored until use at -20°C. Cell treatment was performed at 37°C on human B cells. Cells were plated in 6 well plates at 1.5x106 cells per well and stored at -80°C and total RNA extracted. RTq-PCR was performed according to Genecopoeia. The cycle threshold method (ΔΔCt) was used for data analysis. The comparative Ct method quantification (2^-ΔCt ) and fold change for CD20, CD30, CD40 and CCR5 were - 5.65E+01, 4.80E-01, N/A, 2.47E-01; and 0.954,0.377, N/A and 0.577, respectively. The amount of total RNA extracted from about 4.5x10⁶ cells was low and did not allow us to measure the RNA profile. With the exception of CD40, all other genes were expressed and well-measured in both B cell samples by qRT-PCR. The expression of CD30 and CCR5 genes were decreased in B lymphocytes in vitro with the rose extract treatment. More studies are needed to further study these effects and potential.

Salivary Gene Expression of RANK, RANKL, and OPG in Type 1 Diabetes Mellitus and Periodontal Disease Patients.

The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-β (RANK), receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

Impact of Propolis on Escherichia coli and Bacillus subtilis Based on Total DNA, RNA and Protein Levels.

&lt;b&gt;Background and Objectives:&lt;/b&gt; Propolis is a natural product derived from plant resins and collected by honeybees to protect the colony against different pathogens. The antimicrobial properties of an ethanolic extract of propolis (EEP) were investigated in this study. &lt;b&gt;Materials and Methods:&lt;/b&gt; In the Egyptian Governorate of Giza, F1 carniolan honeybee (&lt;i&gt;Apis mellifera carnica&lt;/i&gt; P.) colonies collected propolis in the autumn, spring and summer of 2019-2020. We investigated the antibacterial activity of EEP against Gram-positive &lt;i&gt;B. subtilis&lt;/i&gt; and Gram-negative &lt;i&gt;E. coli&lt;/i&gt;. Propolis was evaluated and its effects on the amounts of total DNA, RNA and protein in &lt;i&gt;Bacillus subtilis&lt;/i&gt; and &lt;i&gt;Escherichia coli&lt;/i&gt; were investigated. &lt;b&gt;Results:&lt;/b&gt; Propolis inhibited the growth of &lt;i&gt;B. subtilis&lt;/i&gt;. At 5% concentration, the inhibition zones were 8.66, 10.66 and 8.52 mm for autumn, spring and summer propolis, respectively, at 10% concentration, they were 19.66, 21.33 and 18.0 mm, respectively. Propolis also inhibited the growth of &lt;i&gt;E. coli&lt;/i&gt; at a 5% concentration. The inhibition zones were 3.660, 4.00 and 4.33 mm for autumn, spring and summer propolis, respectively and at 10% concentration, they were 7.65, 8.33 and 7.33 mm, respectively. The inhibition zones were the largest (10.66 and 21.33 mm) for &lt;i&gt;B. subtilis&lt;/i&gt; at 5 and 10% propolis, respectively, whereas slightly low inhibition zones (4.0 and 8.33 mm) were observed for &lt;i&gt;E. coli&lt;/i&gt; (5 and 10% propolis, respectively) in spring. In &lt;i&gt;E. coli&lt;/i&gt; and &lt;i&gt;B. subtilis&lt;/i&gt;, propolis inhibited the replication of DNA-dependent RNA polymerase. &lt;b&gt;Conclusion:&lt;/b&gt; The study showed that total DNA, RNA and protein levels in &lt;i&gt;B. subtilis&lt;/i&gt; were reduced by 40, 15.52 and 52.6%, respectively, whereas those in &lt;i&gt;E. coli&lt;/i&gt; were reduced by 12, 15 and 8%, respectively. Furthermore, propolis activity was less effective against Gram-negative bacteria than Gram-positive bacteria.

The effect of Lactobacillus casei and Bacillus coagulans probiotics on liver damage induced by silver nanoparticles and expression of Bax, Bcl2 and Caspase 3 genes in male rats.

In this study, we aimed to evaluate the effect of Bacillus coagulans and Lactobacillus casei probiotics on liver damage induced by silver nanoparticles and expression of Bax, Bcl2 and Caspase 3 genes in rats. 32 adult male Wistar rats were divided into four healthy groups (control), the group receiving silver nanoparticles treated with L. casei, the group receiving silver nanoparticles treated with B. coagulans and the group receiving only silver nanoparticles. The effect of nanoparticles was induced by intraperitoneal injection of silver nanoparticles prepared from nettle at a dose of 50 mg/kg and entered the liver tissue through the bloodstream. Two days after injection, probiotic treatment with 109 CFU was performed by gavage for 30 days. One day after the last gavage, rat liver tissue weight was assessed. Also, the total amount of RNA was extracted from treated, and healthy tissues, as well as induced silver nanoparticles tissues, then evaluated by Real Time PCR. Data were evaluated using one-way Anova, Tukey test. Based on the biochemical results of this study, exposure of rats to different concentrations of silver nanoparticles compared with the control group caused a significant increase in the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), alkaline phosphatase (ALP), especially at high concentrations. Evaluation of damage and histopathological lesions showed that silver nanoparticles in different concentrations caused different damage to liver tissue, so that, necrosis, inflammatory cell infiltration and vascular degeneration were observed at different concentrations by silver nanoparticles. In the present study, the effects of L. casei cell extract on increasing the expression of Bax proapoptotic gene and decreasing Bcl2 gene expression in cancer cells and inducing programmed cell death were shown. In this study, the expression of Bax, Bcl-2 and Caspase-3 genes in the group receiving silver nanoparticles and in the groups treated with probiotics showed significant changes compared to the control group. It can be concluded that the function of silver nanoparticles and the effects of relative improvement of probiotics are from the internal route of apoptosis and factors such as dose, nanoparticle size and nanoparticle coating have an important role in the toxicity of silver nanoparticles, thus the destructive effects on liver tissue could be increased by increasing the concentration of silver nanoparticles.

Maternal age affects equine day 8 embryo gene expression both in trophoblast and inner cell mass

BackgroundBreeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare’s age on blastocysts’ gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed.ResultsMaternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected.ConclusionsThis is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

Improved Methods for Deamination-Based m6A Detection.

N 6-methyladenosine (m6A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m6A has been enabled by the identification of m6A sites transcriptome-wide. However, global m6A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m6A profiling from rare cell types or scarce tissue samples infeasible. To overcome this issue, we previously developed DART-seq, which relies on the expression of a fusion protein consisting of the APOBEC1 cytidine deaminase tethered to the m6A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m6A sites, therefore enabling single nucleotide-resolution m6A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m6A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m6A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m6A detection and will facilitate the mapping of m6A in samples not previously amenable to global m6A profiling.

Molecular spikes: a gold standard for single-cell RNA counting

Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes—RNA spike-ins containing built-in unique molecular identifiers (UMIs) that we use to identify critical experimental and computational conditions for accurate RNA counting in single-cell RNA-sequencing (scRNA-seq). Using molecular spikes, we uncovered impaired RNA counting in methods that were not informative for cellular RNA abundances due to inflated UMI counts. We further leverage molecular spikes to improve estimates of total endogenous RNA amounts in cells, and introduce a strategy to correct experiments with impaired RNA counting. The molecular spikes and the accompanying R package UMIcountR (https://github.com/cziegenhain/UMIcountR) will improve the validation of new methods, better estimate and adjust for cellular mRNA amounts and enable more indepth characterization of RNA counting in scRNA-seq.