A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.

A modification of the TRI Reagent procedure has been elaborated for isolation of RNA from polysaccharide- and proteoglycan-rich material. In the modified procedure, RNA is precipitated from the aqueous phase by the combined action of isopropanol and a high-salt concentration. Under these conditions, RNA is effectively precipitated while contaminating polysaccharides and proteoglycans remain in the soluble form. The modified precipitation does not prolong or increase the complexity of the TRI Reagent procedure. The new procedure was tested by isolation of RNA from polysaccharide- and proteoglycan-rich tissues such as rat liver and aorta.

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.

DNA forms a stable complex with Gene Screen Plus membrane at alkaline pH. Based on this, a method of alkaline transfer of DNA from agarose gel to Gene Screen Plus membrane was elaborated. The procedure entails the use of 0.4 M NaOH for both, the DNA denaturation and DNA transfer steps. The alkaline transfer offers a higher hybridization efficiency and simplifies the transfer procedure as compared with the standard method of DNA transfer at neutral pH. In addition, it can be used to remove RNA contamination from the transferred DNA.

The ratio of absorbance at 260 and 280 nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A260/280 ratio when different sources of water were used to perform the spectrophotometric determinations. Adjusting the pH of water used for spectrophotometric analysis from approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA A260/280 ratios from approximately 1.5 to 2.0. Our studies revealed that changes in both the pH and ionic strength of the spectrophotometric solution influenced the A260/280 ratios. In addition, the ability to detect protein contamination was significantly improved when RNA was spectrophotometrically analyzed in an alkaline solution. UV spectral scans showed that the 260-nm RNA absorbance maximum observed in water was shifted by 2 nm to a lower wavelength when determinations were carried out in Na2HPO4 buffer at a pH of 8.5. We found RNA A260/280 ratios to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 in 1-3 mM Na2HPO4 buffer.

In this report, we present DNAzol, a patent-pending DNA isolation reagent containing guanidine thiocyanate and a detergent mixture. It is a complete, nontoxic and ready-to-use reagent for the isolation of genomic DNA from various biological sources. In the DNAzol protocol, a biological sample is homogenized (or lysed) in DNAzol, and the DNA is precipitated with ethanol, washed and dissolved in 8 mM NaOH. Following pH adjustment, the DNA can be used immediately for analysis or stored at 4 degrees C. The entire isolation can be completed in 20-30 min, and a wide range of DNA molecules can be isolated including genomic DNA and DNA fragments down to 0.1 kb in length. If necessary, samples can be stored in DNAzol at room temperature for extended periods of time. The isolated DNA is ready for PCR, Southern blotting and other molecular biology applications without any additional purification.

Growth hormone (GH) has been implicated in the pathogenesis of proliferative diabetic retinopathy. We sought to determine whether this could be mediated by an effect of GH on proliferation of endothelial cells, and, for this purpose, established long-term cultures of human retinal microvascular endothelial cells (hREC) from normal postmortem human eyes. High-purity (greater than 95%) hREC preparations were selected for experiments, based on immunofluorescence with acetylated low density lipoprotein (LDL) and anti-factor VIII-related antigen. Growth requirements for these cells were complex, including serum for maintenance at slow growth rates and additional mitogens for more rapid proliferation. Exposure of hREC to physiologic doses of human GH (hGH) resulted in 100% greater cell number vs. control (P less than 0.01) but could be elicited only in the presence of serum. When differing serum conditions were compared, hGH stimulated [3H]thymidine incorporation up to 1.6- to 2.2-fold under each condition and increased DNA content significantly in the presence of human, horse, and fetal calf serum. Depending on the culture conditions used, the threshold hGH concentration for significant stimulation of hREC proliferation was 0.4-4 micrograms/liter. In contrast, proliferation of human umbilical vein endothelial cells was not significantly altered by hGH added to concentrations as high as 200 micrograms/liter. In summary, hREC respond to physiologic concentrations of hGH in vitro with enhanced proliferation. This specific effect of GH on retinal microvascular endothelial cells supports the hypothesis of a role for GH in endothelial cell biology.

In the presence of hydrocortisone, plus insulin and prolactin, rat mammary explants transcribe the 25K casein gene, and the half-life of the transcript is about 20 h. The first detectable effect of the withdrawal of glucocorticoid is reduction of the half-life of the casein mRNA to about 1 h, with full retention of the rate of transcription of the casein gene. A later effect of the withdrawal of glucocorticoid is loss of the ability to transcribe the casein gene. Both aberrations can be rectified by hydrocortisone in vitro. The instability of the 25K casein mRNA in the absence of glucocorticoid appears to be related to degradation in the nucleus. Accumulation of actin mRNA in the nuclear fraction is not dependent on glucocorticoid. The results indicate that glucocorticoid exerts a profound, selective effect on the stabilization of rat 25K casein mRNA, and is essential for the transcription of the casein gene. These effects can be dissociated temporally from one another.

The secretion of growth hormone (GH) is controlled by a complex regulatory system. The primary control is exerted by two neuroendocrine hormones, GH-releasing hormone and somatostatin, though other hypothalamic neuropeptides directly and indirectly participate in this process. The regulation of each of these neurohormones is now being clarified at both physiologic and molecular levels, as are their respective roles in the generation of pulsatile GH secretion and in GH feedback regulation. Considerable information has been amassed concerning signal transduction systems mediating the effects of hypothalamic hormones on GH secretion. Although multiple second messengers have been implicated, the adenylate cyclase-cyclic AMP-protein kinase A system appears to exert a predominant role. The developmental regulation of the somatotropes and of GH gene expression is also of importance in determining the GH responses to releasing and inhibiting hormones. The availability of several rodent strains with genetic disorders of growth associated with impaired GH secretion, along with the development of transgenic models, has permitted a more detailed analysis of the role of cytokines and growth factors on both somatotrope biology and hormone secretion. Finally, knowledge gained from studies in animals is permitting a better understanding of the mechanisms involved in physiologic GH secretion and altered GH secretion associated with specific disease states in humans.

We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin is essential for the accumulation of rat casein messenger RNA (mRNA) in the presence of glucocorticoid and prolactin. The accumulation of certain mRNA's in other tissues has also been linked to insulin action. The present study shows that the accumulation effect on the 25,000 molecular weight rat casein mRNA does not reflect stabilization of the transcript by insulin. Rather, insulin is essential for its synthesis in the presence of glucocorticoid and prolactin.

The role of the pituitary, and in particular, of GH in GH-releasing hormone (GRH) gene expression was studied in hypophysectomized rats with and without GH treatment. Hypothalamic GRH mRNA was 6-fold greater in hypophysectomized than in control rats. Increased levels of GRH mRNA were observed at 3 days and the maximal increase was noted at 7 days, postoperatively. Administration of GH to hypox rats partially reversed the increase in GRH mRNA, suggesting a negative feedback regulation by GH of GRH gene activity at the transcript accumulation level. The overall regulation of GRH gene expression, however, appears more complex since GRH mRNA levels and GRH content exhibited discordant changes after both hypophysectomy and GH treatment, suggesting that factors other than GH are required for efficient translation of GRH mRNA.

This report describes a setup for the downward capillary blotting of RNA with the use of 10 x SSC as a transfer solution. The setup is composed of a stack of blotting papers, hybridization membrane, and agarose gel. Two layers of blotting paper connect the stack with two reservoirs containing transfer solution. Using this setup, blotting of RNA fragments (< 7.5 kb) can be completed in 1 h. If necessary, the blotting time can be expanded from 1 to 18 h without decrease in hybridization efficiency of RNA.

This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol BD. In the first procedure, DNA is isolated from 1-20 microL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 microL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.

The role of thyroid hormone and GH in the regulation of hypothalamic GH-releasing hormone (GRH) gene expression in the rat was examined after the induction of thyroid hormone deficiency by thyroidectomy. Thyroidectomy resulted in a time-dependent decrease in hypothalamic GRH content, which was significant by 2 weeks postoperatively, and a reduction in pituitary GH content to 1% of the control level by 4 weeks. In contrast, GRH secretion by incubated hypothalami under both basal and K(+)-stimulated conditions was increased after thyroidectomy. Hypothalamic GRH mRNA levels also exhibited a time-dependent increase, which was significant at 1 week and maximal by 2 weeks after thyroidectomy. Administration of antirat GH serum to thyroidectomized rats resulted in a further increase in GRH mRNA levels. T4 treatment of thyroidectomized rats for 5 days, which also partially restored pituitary GH content, lowered the elevated GRH mRNA levels. However, comparable effects on GRH mRNA levels were observed by rat GH treatment alone. These results suggest that the changes in hypothalamic GRH gene expression after thyroidectomy in the rat are due to the GH deficiency caused by thyroidectomy, rather than a direct effect of thyroid hormone on the hypothalamus, since the changes were reversible by GH alone despite persistent thyroid hormone deficiency. In addition, they further support the role of GH as a physiological negative feedback regulator of GRH gene expression.

Methods presently available for the isolation of RNA are incompatible with conditions necessary for the measurement of either DNA or cell number, resulting in infrequent quantitation of messenger RNA in relation to the quantity of cells studied. In the present studies, a microfluorometric method has been modified from previous techniques to permit the quantitation of DNA in cell lysates prepared using an acid guanidinium thiocyanate-phenol (AGTP) solution from which RNA can subsequently be isolated. The lysate is incubated in alkaline EDTA, then neutralized with KH2PO4, followed by the addition of the fluorochrome bisbenzimidazole (Hoechst 33258), and measurement of fluorescence. DNA content is comparable in measurements by the present technique and by the diphenylamine method on parallel samples. DNA content per cell for human cells measured with this technique is comparable to that previously reported using other methods. The use of AGTP solution results in stability of measurable DNA in cell lysates for periods of at least 10 weeks (permitting batching of samples and retrospective measurement) and stability of fluorescence for at least 20 h after the addition of bisbenzimidazole making the timing of fluorescence measurement less critical. The technique described should permit quantitation of messenger RNA in relation to DNA (and hence indirectly to cell number) on a routine basis.

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA. The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A(+)) mRNAs from total RNA.

Three different methods for RNA preparation using guanidine are presented in this unit--a single-step isolation method employing liquid-phase separation to selectively extract total RNA from tissues and cultured cells and two methods that rely on a CsCl step gradient to isolate total RNA.

It is demonstrated that the accumulation of 42 K casein mRNA in mammary tissue from adrenalectomized, virgin rats is almost 20x higher in the presence of exogenous hydrocortisone than in its absence. Accumulation of 25 K casein mRNA in this tissue is totally dependent on the steroid. The results indicate a much greater dependency on hydrocortisone than was appreciated previously, and also show that this dependency does not reflect a loss of cell viability in the absence of the steroid.

A cell line (GX) derived from a pituitary tumor of a boy with gigantism, which exhibits morphological characteristics of epithelial cells, has been established and maintained in culture for over 2 yr. Initially, GX cells secreted GH which was stimulated by T3. However, after 2 yr in culture, GH production in GX cells is no longer detectable, though GH messenger RNA (mRNA) synthesis has been maintained. The GX cell line is a somatomammotroph line as indicated by the presence of both GH and PRL mRNAs. GX cells were used to study the effect of T3 on GH gene expression. A nearly 3-fold increase in GH mRNA accumulation was observed in GX cells in response to the addition of T3 to the culture medium. This increase was also observed in serum-free medium, indicating that the effect of T3 does not require the presence of other hormones or serum factors. Neither PRL nor beta-actin mRNA accumulation was stimulated by T3 in GX cells. Nuclear runoff experiments demonstrated that the stimulatory effect of T3 on GH mRNA accumulation is related to transcriptional activation of the human GH gene.

Activity of glucocorticoid receptor in mouse mammary cytosol changes during lactogenesis. The highest receptor activity is observed in the second half of pregnancy. The receptor from mammary glands from lactating and pregnant mice exhibits the same sedimentation pattern, as well as the same specificity and affinity for binding of steroid hormones.