Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos.
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[Infectious properties of Rous sarcoma virus RNA and total RNA from tumors induced by this virus].
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Animals↗
A study on postmortem stability of vasopressin messenger RNA in rat brain compared with those in total RNA and ribosomal RNA.
Postmortem stability of arginine-vasopressin (AVP) messenger RNA (mRNA) in the rat brain was studied comparing changes with those in the recovered amounts of total RNA and ribosomal RNA (rRNA). The amount of AVP mRNA and rRNA showed a decrease with increasing time interval after death (postmortem time), whereas the amount of total RNA did not alter with postmortem time. The half-life of AVP mRNA in the rat postmortem seemed to be approximately 16 hrs. The analysis of the ratio of AVP mRNA to 18S-rRNA suggested that AVP mRNA was degraded postmortem more rapidly than rRNA. These results suggest that autopsied human brains should be used for AVP mRNA study within a short postmortem time.
Animals↗
Enrichment of the poly (A) sequence and lack of enhancement of total RNA synthesis in cultured Xenopus hepatocytes by estradiol-17 beta.
The effect of estradiol-17 beta on RNA synthesis and the amounts of total RNA and polyadenylic acid were determined in primary cultures of Xenopus laevis liver parenchymal cells. Results showed that estradiol did not alter the RNA content significantly; control cells contained 11.9 +/- 0.34 micrograms and estradiol-treated cells 12.4 +/- 0.17 micrograms per 10(6) cells on day 2 of estradiol treatment, and 22.0 +/- 0.61 micrograms and 24.0 +/- 1.09 micrograms on day 5. Hybridization with [3H]poly(U) revealed that estradiol increased the poly(A) content about 1.2-fold more than in the controls on day 2 and 1.6-fold on day 5 of estradiol treatment. The actual rate of RNA synthesis was estimated from analyses of the kinetics of [3H]adenosine incorporation into the ATP pool and into RNA. The initial rate of incorporation of ATP into RNA on day 5 of estradiol treatment was 29.38 pmol/min/10(6) cells and the rate of the controls of 29.35. Subsequent accumulation kinetics of [3H]adenosine into RNA showed no difference between estradiol and the control cells. Thus, estradiol did not alter the rate of total RNA synthesis and the total RNA content significantly, but it did increase the poly(A) content.
Adenosine↗
The role of RNA polymerase initiation and elongation in control of total RNA and histone mRNA synthesis in sea urchin embryos.
The involvement of RNA polymerase initiations in regulating total RNA synthesis and the synthesis of the early histone mRNAs was investigated. Nuclei were isolated from developing sea urchin embryos from 4- to 600-cell stages, and the transcription of already initiated polymerase complexes was studied in a "run-off," or elongation, assay; this assay was optimized by using high levels of ribonucleoside triphosphates. Under these conditions the relative levels of RNA synthesis in isolated nuclei from different stages closely paralleled the known rates of synthesis in vivo. However, if sarkosyl is included in the elongation assay, the nuclei of older stages display greatly stimulated synthesis while early cleavage stage nuclei are not stimulated. Sarkosyl does not reveal any elongated transcripts from the early histone genes in nuclei from later stages of development. This has been interpreted to mean that there are many initiated polymerase II complexes that do not elongate rapidly at later stages, but the early histone genes are inactive at later stages because they do not possess any productively initiated polymerases.
Animals↗
Effect of diabetes and insulin treatment of diabetic rats on total RNA, poly(A)+ RNA, and mRNA in skeletal muscle.
We have assessed the time course of alterations in several biochemical parameters and expression of specific mRNAs in gastrocnemius muscle following both the induction of diabetes and the administration of insulin to diabetic rats. Muscle mass, total RNA, and total protein were reduced, whereas poly(A)+ RNA relative to total RNA was increased following the induction of diabetes. All the above parameters, with the exception of poly(A)+ RNA, were reciprocally and rapidly altered following administration of insulin to 3-day diabetic animals. These changes suggest that during the induction of diabetes 1) total cellular protein is reduced at a rate that is less than the reduction in gastrocnemius mass, whereas RNA is reduced at a rate 1.5 times the reduction in tissue mass, and 2) poly(A)+ RNA is elevated relative to total RNA. After insulin administration, there appears to be coordinate synthesis of both poly(A)+ RNA and ribosomal RNA, assuming 85% of total RNA is ribosomal. Therefore, we conclude that poly(A)+ RNA is more stable than ribosomal RNA during diabetes, whereas the amounts of poly(A)+ RNA and ribosomal RNA are increased at the same rates following insulin administration to diabetic animals. Analysis of expression of specific gene products over the same time course, as assessed by in vitro translation of total RNA followed by two-dimensional gel analysis, suggests that there are a few mRNAs that are very rapidly altered in response to insulin administration. The mRNAs that are altered demonstrate variable temporal patterns of either repression or full or transient expression. These rapid, but limited, alterations in gene expression may prove important in the development of the defects that occur in skeletal muscle in response to diabetes.
Animals↗
Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes.
The total RNA content of mouse oocytes, as measured by ethidium bromide fluorescence, was found to decrease by 19% during meiotic maturation (ovulated eggs contain 19% less RNA than full-grown oocytes). Consistent with these results, prelabeled stable RNA of full-grown oocytes decreased by about 20% during in vitro maturation. Polyadenylated RNA represented 19% of total prelabeled RNA in full-grown oocytes and 10% in oocytes matured in vitro, confirming previous results on in vivo prepared material. To distinguish between deadenylation and degradation for one mRNA, the amount and state of adenylation of actin mRNA was examined using Northern blots of oocyte RNA probed with a nick-translated beta-actin cloned chicken cDNA. The results showed that the amount of actin mRNA remained similar during maturation, but its molecular weight decreased slightly. Experiments in which RNA was treated with oligo(dT) and RNase H demonstrated that the actin mRNA was deadenylated during maturation, when actin synthesis is known to decline. These results indicate that the previously defined loss of bulk RNA and changes in the state of adenylation of mRNA during the first 11/2 days of embryogenesis actually begin during the 12 hr of meiotic maturation preceding fertilization.
Actins↗
A micromethod for the isolation of total RNA from adipose tissue.
We have developed a simple and rapid procedure for the isolation of total RNA from small amounts of adipose tissue. Using this method, it is possible to obtain quantitative recovery of RNA from less than 300 mg of adipose tissue, with an average yield of 70 micrograms of RNA per gram of adipose tissue. Northern blot analysis of rat epididymal adipose tissue RNA samples was performed using a beta-actin probe and demonstrated that intact total RNA had been isolated. The procedure has been adapted for use in 1.5-ml microcentrifuge Eppendorf tubes, providing a convenient and inexpensive method for the reproducible recovery of intact RNA from sparse samples of adipose tissue.
Adipose Tissue↗
Total RNA content and blood flow in rat brain after RNA administration.
The changes in blood flow through selected brain structures and the changes in the total RNA content of cells of these structures were examined after a single administration of yeast RNA to 6-month-old male rats. The total content of ribosomal RNA in cells of the limbic system (septum, hippocampus, hypothalamus) increased 48 hrs after the administration of 100 mg i.p. yeast RNA , dropped after 7 days (in hypothalamus), 21 and 30 days (in hippocampus), 30 days (in septum). In cells of the limbic system as a whole there is a higher total RNA content in experimental rats. No changes were observed in the cells of parietal brain cortex. Blood flow increased in limbic structures 21 and 30 days after RNA administration and in septum and in hippocampus also 90 days after application. No changes were observed in parietal brain cortex, bulbi olfactorii, cerebellum and brain stem. Histochemical changes correlated positively with blood flow changes in the limbic system 14, 21, 30 and 90 days after RNA application. The body weight of experimental rats did not differ from that of control animals. The changes in haemodynamic parameters were transient and were demonstrated as fluctuations in heart rate, cardiac output, and peripheral resistance. Blood pressure experienced no changes.
Animals↗
Effects of insulin on total RNA, poly(A)+ RNA, and mRNA in primary cultures of rat hepatocytes.
The purpose of this study was to examine mechanisms involved in the regulation of protein synthesis in primary cultures of rat hepatocytes. Hepatocytes were maintained in a chemically defined serum-free medium in the presence or absence of insulin. The rate of protein synthesis in hepatocytes deprived of insulin between days 2 and 5 of culture was reduced to 67% of the rate observed in insulin-maintained controls. The decrease in protein synthetic rate was accompanied by a proportional fall in the content of both total RNA and poly(A)+RNA, suggesting that the capacity for protein synthesis was reduced in the absence of insulin. Both total RNA and poly(A)+ RNA contents and the protein synthetic rate were returned to control values after 3 days of insulin resupplementation. In addition, the effect of insulin on the expression of specific mRNAs was assessed by in vitro translation of total RNA followed by two-dimensional gel analysis of radiolabeled translation products. Only 13 of the greater than 150 spots discernible on the two-dimensional gels were altered in response to insulin. The mRNAs that were altered include examples of repression and stimulation of expression in response to insulin deprivation. Thus, in isolated rat hepatocytes, insulin regulates the capacity of both overall protein synthesis as well as the capacity for the synthesis of specific proteins.
Animals↗
Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.
A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.
Artifacts↗
[Effects of dibutyryl derivatives of cyclic nucleotides on synthesis of total RNA and proteins in cultured fetal rat hepatocytes].
During short-term (6 h) or long-term (24 h) incubation of fetal rat liver cells in primary cultures, 10(-3) M dibutyryl-derivative of cyclic AMP (Bt2cAMP) and sodium butyrate decreased total RNA and protein synthesis. In contrast, dibutyryl-cyclic GMP (Bt2cGMP) at the same dose (10(-3) M) was without significant effect on RNA and protein biosynthesis. During short-term (4 h) incubation 10(-3) M Bt2cAMP and Bt2cGMP stimulated serum albumin production, while sodium butyrate was without effect. In long-term (22 h) incubation only 10(-3) M Bt2cAMP noticeably increased albumin production. The results obtained clearly show that Bt2cGMP, unlike Bt2cAMP, is not able to modify significantly total RNA and protein synthesis in cultured fetal rat liver cells. It is concluded also that the effects of dibutyryl-derivatives of cyclic nucleotides, at least on albumin production, are not mimiebet by butyrate.
Animals↗
An analysis of total RNA translation products of rat liver during regeneration with a comparison to fetal liver.
We explored differences in the mRNA populations of regenerating, sham-operated, and fetal rat liver using two-dimensional gel electrophoresis to resolve the radiolabeled protein products of liver total RNA translated in vitro. Twenty-four translation products were changed significantly after partial hepatectomy and sham hepatectomy. Nine of the 24 products changed after partial hepatectomy only, while the remainder also changed after sham hepatectomy. Two of the nine increased during regeneration to relative levels similar to those found in fetal liver. However, substantial differences also exist between the translation products of regenerating and fetal liver. These results suggest that the response to partial hepatectomy involves the alteration of mRNAs present in normal liver and that this response does not duplicate the fetal pattern of hepatic total RNA translation activity.
Animals↗
Rapid isolation of total RNA from mammalian tissues.
A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.
Animals↗
[Hormonal regulation of total RNA and protein biosynthesis in liver cell cultures of rats in the pre- and postnatal period of development].
The effects of several hormones on total RNA and protein biosynthesis were examined in primary cultures of liver cells obtained from rat fetuses on 21-22 days of gestation and from 3 week-old weanling rats. The intensity of biosynthesis processes was estimated by the incorporation of labeled precursors in macromolecules. Insulin, cortisol, triiodothyronine (T3) stimulated RNA and protein biosynthesis in both types of cultures. These hormones enhanced total protein biosynthesis in fetal rat liver cells more efficiently than in hepatocytes of weanling rats. Somatotropin (growth hormone--GH) did not change total protein biosynthesis but notably increased RNA synthesis and the production of immunoreactive serum albumin. Experiments on fetal rat liver cell cultures showed that stimulating action of cortisol on RNA synthesis was synergistic in relation to the effects of insulin and GH. It has been concluded that fetal rat liver cell at the end gestation are able to respond adequately to anabolic action of the hormones.
Animals↗
Spatial Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Tissue by spRandom-seq.
The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.
Paraffin Embedding↗
A social environment influences the total RNA content of the brain cells of young laboratory rats.
The paper describes the results of measurement of the total RNA content of single cells of the frontal pole of the cerebral cortex, the septum, the dorsal hippocampus and the medial hypothalamus of 35-day-old male laboratory (Wistar strain) rats. Two groups of ten animals each, reared in different ways, are compared: 1) young which lived from the age of 15 days with their mother, their siblings, other adult animals of both sexes and the young of other litters in a large, complex space, and 2) young which lived only with their mother and siblings in a small, simple space, as is usual under laboratory conditions. Nutrition and other environmental factors in the two groups were the same. The RNA content of cells from all the given parts of the brain was higher in community-reared rats, but the relative difference (related to the mean values for community-reared animals) in the various parts was not the same. It was smallest in the cerebral cortex and greatest in the dorsal hippocampus. Differences in the ventromedial hypothalamus and the septum were moderately large, with the hypothalamus rather resembling the cerebral cortex, while the septum differed statistically significantly from both the cortex and the hippocampus. The effect of social stimulation was thus strongest in the hippocampus.
Animals↗
Cultured human keratinocytes: discrimination of different cell cycle compartments based upon measurement of nuclear RNA or total cellular RNA content.
Correlated measurements of total cellular RNA and DNA of cultured human keratinocytes by flow cytometry, followed by multivariate analysis, discriminate three distinct subpopulations of cells differing in RNA content. The first subpopulation is comprised of small cells resembling basal cells of epidermis, with low RNA content and long (100-300 h) generation times. The second subpopulation consists of keratinocytes resembling cells in the spinous layer of epidermis, characterized by increased RNA content and shorter (35-40 h) generation times. The third subpopulation consists of the largest, keratinohyalin-containing cells which remain in G1 and undergo terminal differentiation. In contrast to total cellular RNA, correlated measurements of DNA and nuclear RNA reveal that: (1) entrance of all cultured cells from G1 into S phase occurs only after accumulation of the same, threshold amount of nuclear RNA; hence there is only a single population of S + G2 + M-phase cells; (2) there are two distinct subpopulations in G1, one with minimal nuclear RNA content and another with increased RNA. Stathmokinetic experiments indicate that the G1-phase cells with low nuclear RNA have distinctly longer residence times in G1 compared to cells with high nuclear RNA content. Thus, measurements of the total cellular RNA versus nuclear RNA content reveal kinetically distinct cell subpopulations. Whereas total cellular RNA content correlates more with differentiation, nuclear RNA content reflects primarily the kinetic properties of the cell.
Acridine Orange↗