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PubMed1999

Isolation of total RNA from dermatophytes.

We report a method for the preparation of total RNA from the anthropophilic dermatophyte Trichophyton rubrum. To generate large quantities of mycelia, the fungus was grown in liquid culture medium. The harvested mycelial mass was ground to a fine powder in liquid nitrogen and homogenized in guanidine isothiocyanate buffer followed by ultracentrifugation of the obtained suspension through a caesium chloride gradient. Analysis of the prepared RNA showed two prominent ribosomal RNA (rRNA) bands of about 3.36 and 1.82 kb. Northern blot hybridization with a beta-actin cDNA confirmed the high quality of the fungal mRNA. Successful isolation of RNA from two other dermatophyte species, namely Trichophyton mentagrophytes and Microsporum canis, demonstrated the general applicability of the described procedure.

Humans↗
PubMed2003

Molecular assay to generate expression profile of eight estrogen receptor alpha isoform mRNA copy numbers in picogram amounts of total RNA from breast cancer tissues.

The presence of estrogen receptor alpha (ERalpha) is the basis for treating breast cancer patients with targeted molecular therapies that block estrogen stimulation of breast cancer cell division. Currently, the ERalpha presence is determined by microscopically scoring the slides subjected to immunohistochemistry. This method is not quantitative, cannot distinguish between all the known ERalpha isoforms, and requires large amounts of tumor tissue. We describe here a real-time PCR-based molecular approach that can be applied to determine mRNA copies of eight ERalpha isoforms in picogram amounts of total RNA from clinical samples. Each isoform mRNA is quantified using a specific primer pair and a 5'FAM- and 3'TAMARA-labeled probe in comparison with a standard curve constructed with known copy numbers of its respective reverse-transcribed cRNA. Seven alternatively spliced isoforms were quantified using splice-targeted primers. The cRNAs for eight isoforms were generated by in vitro transcription of their respective coding sequences. The sensitivity of detection with reverse-transcribed cRNAs is as low as 100 copies. The devised assays can detect ERalpha cDNAs reverse transcribed from as low as 50-100 pg of total RNA from breast cancer tissues. The applicability of the devised assays for profiling eight ERalpha isoform mRNAs is demonstrated using 6 breast cancer cell lines and 10 breast cancer tissues. It is expected that these assays could be applied to profile ERalpha isoforms in any estrogen-responsive tissues. In addition, these methods could highly facilitate the design of tissue-specific selective estrogen receptor modulators to treat breast cancers and other estrogen-related abnormalities.

Alternative Splicing↗
PubMedDate not supplied

[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↗
PubMed2003

[A simple and effective method for extracting total RNA from domestic fungus].

With Flammulina velutipes material,an improved method was developed for extracting total RNA from domestic fungus that are rich in RNase,polyphenols, polymeric carbohydrates and proteoglycans. Phenol-chloroform-isoamyl alcohol were used twice to clear DNA and protein under higher concentration of denaturing solution and isopentanol, sodium acetate were used to precipitate RNA selectively. Pure and intact RNA can be effectively prepared by this method.

English Abstract↗
PubMed2026

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↗
PubMed2001

An effective, rapid and simple method for total RNA extraction from bacteria and yeast.

In this work, we describe a rapid and simple method for total RNA extraction from bacteria and yeast. The method allows for the acquirement of high RNA yields while avoiding the use of phenol or other toxic reagents and is less expensive than other methods previously described. The extracted RNA is suitable for applications such as RT-PCR, Northern blot hybridization and low molecular weight RNA (LMW RNA) electrophoresis.

Bacteria↗
PubMed2003

Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate.

Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.

Ascorbic Acid↗
PubMed2003

Total RNA suitable for molecular biology analysis.

Development of methods based on determining expression of individual genes resulted in the need for large amounts of high quality RNA preparations. It is widely accepted that in intact rRNA the 28S and 18S band ratio must be 2:1. It is not quite clear what is the main cause of lower rRNA bands intensity ratio. It is difficult to isolate RNA with 2:1 28S/18S ratio from RNase-rich and some tumor tissues. At the same time this requirement may be excessive and RNA preparations with lower 28S/18S rRNA ratio may be quite adequate for most techniques of determining gene expression. As demonstrated in this study, the level of a particular RNA may be reliably determined by RT-PCR even in a total RNA that is usually considered as degraded (28S to 18S ratio as low as 0.4), provided that random primer is used in RT. In contrast, the use of the oligo(dT) primer in RT-PCR may lead to underestimation of specific mRNA level in the degraded RNA samples, depending on the distance of amplified fragment from the poly(A) end. A criterion based on average degradation level of a number of reference genes is suggested to discriminate specific RNA degradation from random and unspecific ones.

Animals↗
PubMed2003

Improved method for generating microarray probes using submicrogram amounts of total RNA.

The ability to identify disease-associated genes using microarray technology is dependent on isolating high-quality total RNA from the diseased tissue under study. However, it is not always possible to obtain large amounts of affected tissue from patients and therefore the quality and yield of RNA may be compromised. This protocol described a procedure for generating superior microarray probes by amplifying RNA sequences via successive rounds of in vitro transcription (IVT) reactions. This improved procedure utilizes a 9-mer primer to generate the IVT template, which is able to recapitulate the size ditribution of the original isolated RNA sample.

Algorithms↗
PubMed1984

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↗
PubMed1989

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↗
PubMed1999

Validation of a rapid method for extraction of total RNA applied to Leishmania promastigotes.

This report explains a rapid procedure (approximately 50 min) for the isolation of highly purified total RNA from Leishmania promastigotes based on extraction with acidic phenol. The simplicity of the manipulations required make this method ideal for processing multiple samples; the quality of the RNA obtained is suitable for reverse transcription polymerase chain reaction analysis.

Animals↗
PubMed2003

[A novel method of extracting total RNA from rice embryo samples].

A novel and simple CTAB-LiCl-based extraction method for high-quality and total RNA of rice embryo samples is developed. This method can efficiently eliminate the interference of polysaccharide and lipids rich in rice (Oryza sativa L.) embryo obtained under room temperature without using liquid nitrogen. The results of ultraviolet spectrophotometer and agarose gel electrophoresis analysis show that the obtained RNA has no obvious degradation and a good purity sufficient for further RT-PCR and RNA gel blotting. Therefore, it is also especially useful for the RNA extraction of plant material plenty of polysaccharide and lipids.

English Abstract↗
PubMed2001

[The analysis of rice total RNA by capillary electrophoresis].

When the RNase was eliminated through backing at 200 degrees C or treated by diethylpyrocarbonate (DEPC), the total RNA of rice could be separated within 15 min using 1.0%T, 0%C linear polyacrylamide as sieving matrix and 7 mol/L urea as denaturant. The tRNA of rice can be separated into two classes and about nine peaks when high concentrated polyacrylamide sieving matrix (5.0%T, 0%C) was used. This technique could provide one of the rapid and accurate methods for the determination of RNA in plants.

English Abstract↗
PubMed1998

Purification of total RNA from human stool samples.

While colonoscopy may detect early-stage colon tumors, a less invasive and more cost-effective technique would be beneficial. Stool, which picks up sloughed-off colonic epithelial cells, would be ideal for sampling the mucosa; shed tumor cells may display alterations in gene expression observed in intact tumors. It is first necessary, however, to show that RNA can be isolated from human feces and that this RNA contains human gene transcripts. We have therefore developed a method for the isolation of total RNA from freshly passed human stool, consisting of lysis in chaotropic agents, repeated extraction with phenol and phenol-chloroform, and absorption with an RNA-binding resin. After treatment with RNase-free DNase I, we assayed these preparations for the presence of human RNA by quantitative slot blotting, northern blotting, and reverse transcription-polymerase chain reaction (RT-PCR). We obtained 5-30 microg RNA per gram of stool from cancer patients, and about 5 microg RNA per gram of control stool. Quantitative slot blotting showed that about 10% of this RNA was of human origin. Both northern blotting and RT-PCR demonstrated the presence of human RNA in these samples. To unambiguously demonstrate the isolation of RNA from stool, we incubated a mixture of rat cells and control human stool at 37 degrees C for up to 24 hr. RT-PCR of the RNA isolated from this sample clearly revealed the presence of rat-specific mRNA. These experiments indicate that RNA can be isolated from human stool and that message encoded by human genes can be assayed in these preparations. This procedure may provide a powerful tool to identify patients at risk for colon cancer.

Animals↗
PubMed1995

Two rapid microscale procedures for isolation of total RNA from leaves rich in polyphenols and polysaccharides: application for sensitive detection of grapevine viroids.

Two rapid microscale procedures for isolation of total RNA from grapevine leaves are described. Homogenized leaf tissue is phenol extracted and polyphenols and polysaccharides are removed by either an aqueous two-phase system followed by DEAE-cellulose chromatography or by differential precipitation with the solvent 2-butoxyethanol. The resulting RNA, about 5-15 micrograms from 0.25 g grapevine leaves, is of high quality and can be employed for reverse transcription and polymerase chain reaction (PCR) amplification or Northern blot analysis to detect grapevine viroids or other RNAs. The two procedures are optimized for small-scale examination of many samples in a short time. Thus RNA from 12-24 leaf samples can be analyzed per day, including reverse transcription and PCR amplification. The application of these procedures led to the first detection of grapevine yellow speckle viroid 1 in German grapevines.

Base Sequence↗
PubMed2004

High-accuracy amplification of nanogram total RNA amounts for gene profiling.

Microarray-based gene profiling of laser-assisted microdissected tissues or clinical biopsies is still a challenge since the amount of total RNA in such samples is limited and amplification of RNA is mandatory. Representative amplification of mRNA is highly dependent on the reverse transcription reaction, which is error prone, and on the number of amplification cycles. To improve the accuracy of RNA amplification, we optimized, combined, and tested different amplification strategies for Affymetrix oligonucleotide array hybridization. We demonstrate that different protocols differ significantly in quality of mRNA amplification. To demonstrate the accuracy and reproducibility of our optimized protocol in a clinical setting, we analyzed total RNAs from laser-assisted, microdissected cells of human prostate tissues. On the basis of these results, we recommend a standard reverse transcription reaction for small-sample-transcriptome profiling experiments as part of the Minimal Information about a Microarray Experiment (MIAME) set of standards.

Animals↗
PubMed1989

Rapid isolation of total RNA from small mammal and human skeletal muscle.

The present study was designed to assess the efficacy and reliability of a method (Anal. Biochem. 162: 156-59, 1987) of total RNA isolation from small muscle samples. The RNA content of skeletal muscles in mouse, rabbit, guinea pig, rat, and human was also investigated. Enough RNA was extractable from muscle samples as small as 25 mg in weight to provide material for the analysis of specific mRNA sequences using RNA blot hybridization with a cDNA probe for a mitochondrially encoded subunit of cytochrome-c oxidase. The RNA was free of detectable levels of protein or DNA. Gel electrophoresis and cDNA probe hybridization indicated that the RNA was relatively undegraded. Significant differences (P less than 0.05) in RNA content were found among fiber types (type I fibers greater than type II fibers) and among the soleus muscles of various species (guinea pig greater than rat = mouse greater than rabbit). Human muscle possessed the lowest RNA content (350 ng/mg) among the species investigated. The results indicate that intact RNA can be rapidly and reliably isolated from small skeletal muscle, including samples typically obtained from human muscle biopsies. This technique should permit the analysis of gene expression in muscle subject to a variety of physiological treatments.

Animals↗