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PubMed1990

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↗
PubMed1979

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↗
PubMed2000

Extraction of total RNA from a high pigment content plant. Marigold (Tagetes erecta).

The Mexican marigold (Tagetes erecta) produces inflorescences of intense yellow color that contain high levels of xanthophylls, particularly lutein, which makes it a suitable model for the study of carotenoid biosynthesis and regulation throughout the development of the inflorescences. However, these studies require the recovery of total RNA from floral buds and inflorescences at different developmental stages, each of which presents specific extraction problems. Four protocols were tested, but only through the modification of one of them was it possible to obtain total RNA of sufficient quality and quantity to perform RT-PCR and Northern blots and to construct a cDNA library. This article presents the modified protocol for the recovery of total RNA from carotenoid-rich plant tissues.

Asteraceae↗
PubMed1992

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↗
PubMed1998

Rapid method for isolation of total RNA from eukaryotic cell lines and leukocytes.

Total RNA was isolated from human leukocytes (monocytes, granulocytes), various cell lines (COS-7, Mono-Mac-6, L-132, HaCaT, EA.hy926, HL-60), and fungal mycelium by a rapid two-step method. Cells were lysed with NaDodSO4 in a citric acid-containing buffer. This procedure was succeeded by salt precipitation to remove contaminating DNA and protein and a final alcohol precipitation of RNA. Isolated RNA was of high quality, with a reasonable yield and little or no protein or DNA contamination. We present here a fast method for preparing RNA particularly from cell lines, which limits the use of toxic compounds.

Animals↗
PubMed2001

Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays.

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.

Cell Extracts↗
PubMed1995

A simple and efficient method for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe.

A simple and efficient procedure was described for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe. The present study demonstrated that the quality and the quantity of S. pombe RNA were increased by substituting phenol/chloroform mixture for phenol as a deproteinizing agent in the first vortexing step and using an ice bath instead of a dry ice-ethanol bath in the freezing step. Additionally, this protocol had the advantage of extracting total RNA without any degradation of S. pombe cells. Furthermore, the high amounts and quality of RNA extracted by this modified procedure enabled us to perform some experiments such as Northern blot, S1 mapping, primer extension, and reverse transcriptase reaction-polymerase chain reaction (RT-PCR) without further RNA purification. We suggest that this procedure is very useful to analyse primary structures and steady-state levels of RNA from S. pombe.

Base Sequence↗
PubMed1997

Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect.

Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A) RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.

DNA, Complementary↗
PubMed2002

Isolation of total-RNA from formalin-fixed rat retina.

The aim of this project was to establish a method for the purification of total-RNA from fixed rat-retina. Two different established methods were used for RNA purification, and successful isolation was verified with RT-PCR for amplification of beta-actin (two different product-lengths) and subsequent gel-electrophoresis. Total-RNA was successfully isolated from fixed rat-retina. The house keeping gene, beta-actin could be detected after fixing the retina either with 1% formalin or with 4% paraformaldehyde (PFA). Hexamer-primer based RT-PCR gave better results than the oligo-d(T)-primer based RT-PCR method. Both the 698 and 225 bp beta-actin-fragments could be successfully amplified, where amplification of the latter was more efficient. This approach shows that tissue fixation prior to RNA-isolation facilitates the rapid isolation of undamaged RNAs in tissues such as the retina, which are known to yield low levels of RNA and are vulnerable to RNases.

Animals↗
PubMed1997

Amplification of a full-length Borna disease virus (BDV) cDNA from total RNA of cells persistently infected with BDV.

We have developed a novel reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, corresponding to the 5'-terminus of a putative 3'-leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53-mer antigenomic primer and a 25-mer primer, corresponding to the 3'-terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof-reading activity. The amplified full-length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT-PCR method should be a useful technique to study the molecular quasispecies of BDV.

Animals↗
PubMed2005

[Induction of efficient T-cell immunity against autologous leukemia cells by dendritic cells pulsed with the leukemia cell total RNA].

OBJECTIVE: To assess the feasibility and efficiency of eliciting leukemia-specific T cell responses in acute myeloid leukemia patients in complete remission (AML-CR) in vitro by dendritic cells (DC) pulsed with the leukemia cells total RNA. METHODS: The immature DCs were generated from the adherent bone marrow mononuclear cell in vitro in the presence of combined cytokines (GM-CSF 100 ng/ml, IL-4 500 U/ml), and pulsed with total RNA isolated from autologous leukemic cells by cationic lipid 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP) at day 5 of culture. Then the cells were incubated for another 24 h in a medium containing 10 ng/ml of TNF-alpha for maturation of DC. After the total 7 days culture, the cells were harvested as the mRNA-DC and the expression of mature DC markers were determined by FACS. The proliferative capacity of T cell activated by mRNA-DC was determined by MTT assay. Meanwhile, the mRNA-DC was co-cultured with T lymphocytes at a ratio of 1:3 for 7 days. The activated T lymphocytes were harvested, the secretion of IFN-gamma was determined by ELISPOT assay, and the cytotoxicity was analyzed in vitro by LDH release assay. RESULTS: After culture, the BMMNC from 14 AML-CR patients developed morphologic and phenotypic characteristics of mature DC. At a stimulator/reactor ratio of 1:16, auto-T lymphocytes primed with mRNA-DC exhibited significant proliferative activity compared with T lymphocyte primed with non-pulsed DC [(36.84 +/- 5.68)% vs (12.20 +/- 3.16)%, (P < 0.05)]. An expansion of mRNA reacted T cell secreting IFN-gamma could be observed on ELISPOT assay. At an effector/target ratio of 20:1, the auto-T lymphocytes primed with mRNA-DC exhibited significant killing activity to auto-AML cells (45.46 +/- 6.34 )% as compared with that stimulated by IL-2 alone (13.26 +/- 2.28)% or primed by non-pulsed DC (12.32 +/- 1.32)% (P < 0.05). CONCLUSION: Immunization with DC-leukemia cell RNA vaccines may be a simple, rapid and potent approach to elicitation of T cell-mediated anti-leukemia immunity.

Adolescent↗
PubMed1992

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↗
PubMed2002

Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis.

Fine-needle aspirate samples hold the potential for gaining valuable insight into the molecular details and prognostic indicators for certain types of cancer in a limited volume of relatively pure tumor cells. Although limited, such clinical samples can be used with high efficiency when analyzed in conjunction with gene-dense expression microarrays. For this reason, it is essential to retrieve as much high-quality genetic material as possible from each fine-needle aspirate sample. We have conducted a study to improve the efficiency of extracting high quality total RNA to use in microarray analysis from single ex vivo fine-needle aspirate samples of 11 breast cancers added to RNAlater RNA Stabilization Reagent immediately upon collection. Approximately half the total RNA from fine-needle aspirate samples of breast cancers was isolated from the supernatant, and that RNA had similar quality and gene expression profile to the RNA that was isolated from the corresponding cell pellet. We recommend that the supernatant not be discarded when extracting RNA from fine-needle aspirate samples stored in RNAlater.

Biopsy, Needle↗
PubMed1991

[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↗
PubMed1999

A calcium homeostasis mechanism induced by heterologous expression of total RNA from chicory leaves in Xenopus oocytes.

Xenopus oocytes were injected with total RNA from chicory leaf tissues and then examined by the voltage-clamp technique. A double-step voltage protocol was used, with an initial hyperpolarization step from the holding potential of -35 to -140 mV followed by a second depolarization step to +60 mV. Two different outward currents were observed, one noninactivating (Ini), and one inactivating (Ii). Only the noninactivating outward current (Ini) could be induced by depolarization from -35 to +60 mV. The mean amplitude of Ini was 2915 +/- 848 nA (n = 11). This current, carried by chloride ions, declined nearly to the baseline in 153 +/- 64 sec (n = 13), and was highly dependent on intracellular calcium. After the rundown of Ini, the same oocyte was depolarized from -140 to +60 mV. This protocol induced an inactivating outward current (Ii) with a mean amplitude of 4461 +/- 1605 nA (n = 13). Ii was also carried by chloride ions and dependent on extracellular calcium. Ii was strongly inhibited by 100 micron extracellular La3+. These two types of chloride currents were also observed after IP3 injection in control oocytes. Ini and Ii were not observed in noninjected oocytes or water-injected oocytes. We suggest that the expression of total chicory leaf tissue RNA in Xenopus oocytes reveals a calcium homeostasis mechanism responsible for calcium mobilization from internal stores and subsequent calcium entry.

Animals↗
PubMed1989

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↗
PubMed1996

[Repression of voltage gated K+ channel expressed in Xenopus oocytes after injection total RNA from carp retina by an antisense oligonucleotide].

Injection of total RNA from carp retina induced the formation of a membrane K+ channel in Xenopus oocytes. We found an oligonucleotide complementary to the mRNA coding for a peptide fragment in the K+ channel of drosophila and mice could suppress the K+ channel current in a very specific manner. This provides important information for the cloning of this gene and for studying it's function.

Animals↗
PubMed1988

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↗