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At least 127 records · Page 7Linked to original sources
PubMed1991

A sensitive RNase protection assay for the quantitation of the mRNAs for the LDL receptor and HMG-CoA reductase in human total RNA. Effects of treatments on cells in culture designed to up- and down-regulate expression of the LDL receptor.

Regulation of expression of the genes for the low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is of central importance in the control of cholesterol metabolism and thus in influencing the concentration of low density lipoprotein in the plasma. This can be studied by investigating the effects of factors (hormones, drugs, etc.) on the levels of mRNA for these genes. An RNase protection assay is reported for measurement of the levels of mRNA for the LDLR and HMGR. Several probes have been developed for these genes, together with probes for the "housekeeping" genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Various conditions in the assay have been examined and optimised, e.g. conditions for solution hybridization and RNase digestion and the use of "sense" RNA standards. The assay allows accurate measurement of approximately 2 x 10(7) copies of LDLR and HMGR mRNAs, which is equivalent to the number of copies present in approximately 1 x 10(6) human dermal fibroblasts and approximately 5 x 10(5) Hep G2 liver hepatoma cells cultured in 10% fetal calf serum. The average number of copies of mRNA per cell was estimated in fibroblasts and Hep G2 cells under various conditions of regulation of the LDLR and revealed the following: [table: see text] Under the chosen conditions 10 copies per cell was the detection limit for the assay. The effect of these treatments on the number of copies of mRNA per cell for beta-actin and glyceraldehyde-3-phosphate dehydrogenase was also determined.

Actins↗
PubMed1995

Simultaneous extraction of total RNA and peptides from tissues: application to tachykinins.

Methods for extraction and isolation of intact RNA are often laborious, time consuming, and preclude the direct analysis of peptides. Similarly, the conditions for extraction and isolation of peptides are unsuitable for the isolation of intact RNA. Thus, to study changes in the levels of neuropeptides and gene expression of the corresponding mRNAs, separate procedures are required. A simple and rapid method for the simultaneous extraction of RNA and peptides from tissues is described. RNA and peptides are extracted with guanidinium isothiocyanate, followed by delipidation, and peptides are isolated by a simple solid-phase extraction procedure. RNA is isolated by differentially partitioning DNA into an organic phase, followed by precipitation with ethanol. The RNA and peptides isolated by this method are of high yield and quality. Furthermore, this method for RNA isolation is successful and efficient, even with tissues that proved recalcitrant with other procedures, and allows the simultaneous processing of multiple samples. We describe the successful application of this procedure for measuring tachykinins and the corresponding preprotachykinin A mRNA from tissues. Extraction of neuropeptide K, a 36-mer tachykinin, was dramatically more efficient with the present method than other methods in common use.

Animals↗
PubMed1997

Quantification of total RNA by ethidium bromide fluorescence may not accurately reflect the RNA mass.

The fluorescent signal from purified large and small ribosomal RNA subunits stained with ethidium bromide was quantified using agarose gels and image analysis. A significantly smaller fluorescent signal was observed for 23 S rRNA compared to 16 S rRNA when normalized to mass. Therefore, small changes in amounts of 23 S or 16 S rRNA within a sample can have a profound impact on observed bulk fluorescence. A comparison of 23 S/16 S rRNA fluorescence ratios for 4 marine bacteria grown in batch culture indicates that the lower fluorescence of 23 S rRNA is widespread. The 23 S/16 S rRNA fluorescence ratios also suggest that intracellular concentrations of ribosomal subunits do not always adhere to a stoichiometry of 2.0.

Ethidium↗
PubMed1998

Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage.

The stability of RNA, particularly mRNA, in tissues is under complex regulation. Most studies to date have focused on very cellular tissues and not connective tissues such as ligaments, tendons and cartilage. As the availability of such tissues for transplantation or research purposes is frequently delayed following death, it is important to determine whether RNA stability in such tissues is influenced by time postmortem. To approach this question, skeletally mature NZW rabbits were used to investigate RNA integrity over time in dense, hypocellular connective tissues and in several hypercellular organ tissues such as brain, kidney, liver and lung. Samples were analyzed at varying intervals postmortem with respect to rRNA integrity by agarose gel electrophoresis and ethidium bromide staining and mRNA integrity by Northern blot analysis and RT-PCR. No degradation of rRNA or loss in integrity of mRNA for genes of low and high copy number was observed up to 96 h postmortem. These findings confirm that it is likely appropriate to use properly stored postmortem dense connective tissues for molecular biological investigations.

Animals↗
PubMed1991

Polyinosinic acid as a carrier in the microscale purification of total RNA.

Three different RNA carriers were compared for use in microscale RNA isolation and subsequent cDNA synthesis and amplification via the polymerase chain reaction. E.coli rRNA alone gave considerable cDNA synthesis which under standard carrier conditions overwhelmed cDNA synthesis from lymphocyte mRNA. Yeast tRNA caused inhibition of mRNA primed cDNA synthesis, giving low levels of cDNA synthesis when used without cellular RNA. In contrast, commercially available poly I alone did not prime detectable cDNA synthesis nor did it inhibit such synthesis primed by cellular mRNA. When RNA preparations were made using these three carriers and decreasing numbers of starting lymphocytes, poly I allowed the detection of cDNA from two orders of magnitude fewer lymphocytes than the other carriers. Thus poly I was found to be a superior carrier molecule for microscale RNA preparations suitable for reverse transcription and subsequent amplification using the polymerase chain reaction.

Animals↗
PubMed1968

Effect of the kinetin-naphthaleneacetic acid interaction upon total RNA and protein in senescing detached leaves.

The interaction between kinetin and naphthaleneacetic acid in the regulation of senescence of excised tissue of mature broccoli leaves has been used to examine the extent of synchrony between changes in chlorophyll, RNA, and protein. Kinetin increased the net uptake of (14)C-labeled orotic acid and leucine. Naphthaleneacetic acid decreased the effect of kinetin on net uptake after long treatment, but in short-time treatments the auxin increased the effect of kinetin on net uptake. Results of long (24 hr) treatments indicated a general synchrony between the loss of RNA, protein, and chlorophyll. Naphthaleneacetic acid reduced the stabilizing effect of kinetin upon chlorophyll content and upon the content and synthesis of RNA. In short-time experiments, however, RNA content and synthesis were transiently increased by kinetin, and further increased by kinetin plus naphthaleneacetic acid, while chlorophyll content decreased in the presence of kinetin and decreased further in the presence of kinetin plus naphthaleneacetic acid. Actinomycin-D accelerated the loss of chlorophyll, RNA and protein and strongly depressed the rate of RNA synthesis. In the presence of actinomycin-D the stabilizing effect of kinetin upon RNA was substantially reduced. In contrast, the chlorophyll and protein contents remained higher than in the control. Actinomycin-D did not nullify the basal incorporation of orotic acid into RNA, nor did it negate the effect of kinetin upon incorporation. The failure of synchrony between changes in chlorophyll and RNA does not substantiate the proposal that kinetin regulates senescence by a direct effect upon DNA-dependent RNA synthesis.

Naphthaleneacetic Acids↗