Analysis of RNA by northern blot hybridization.

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. The Basic Protocol describes blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support protocol.

Analysis of RNA by northern and slot-blot hybridization.

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting), while unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Included in this unit are detailed procedures for RNA denaturation, blotting and hybridization. Also described is a method for stripped hybridization probes from blots so the blots can be re-hybridized with a different probe.

Experiences of older women with cancer receiving hospice care: significance for physical therapy.

BACKGROUND AND PURPOSE: The number of older adults with cancer is growing, increasing the need for professionals who are able to meet these patients' special needs. In palliative care settings, physical therapists strive to promote quality of life. Minimal research exists, however, to guide therapists working with patients with terminal illness. The purpose of this study was to gain knowledge that can be used by physical therapists to more effectively assess and treat older people with cancer receiving hospice care. SUBJECTS AND METHODS: A qualitative single-case study with replication was conducted with 3 older women with cancer who were receiving hospice care. Interview data were analyzed using grounded theory techniques. RESULTS: Four themes emerged as central to the experience of the informants: social relationships, spirituality, outlook on mortality, and meaningful physical activity. CONCLUSION AND DISCUSSION: In addition to maintaining physical function, physical therapists, who attend to nonphysical as well as physical aspects of care, may foster social cohesion, help maximize life's meaning, and support stabilizing strategies of older women with cancer who receive hospice care.

Loss of heterozygosity of the long arm of chromosome 7 in follicular and anaplastic thyroid cancer, but not in papillary thyroid cancer.

Papillary thyroid cancer (PTC), but neither the follicular nor the anaplastic histotype [follicular thyroid cancer (FTC), anaplastic thyroid cancer (ATC)], overexpresses simultaneously the protooncogene HGF (hepatocyte growth factor) and its receptor HGF-R (or c-met). Because 1) HGF and c-met map to chromosome 7q21 and 7q31, respectively, 2) FTC loses genetic material at multiple loci with a frequency much higher than PTC, and 3) loss of heterozygosity (LOH) on 7q has been previously found in various tumors, we tested the hypothesis that both FTC and ATC, but not PTC, could harbor LOH in segments of 7q encompassing the loci for HGF and c-met. We screened 6 normal thyroids, 10 colloid nodules, 10 follicular hyperplasias, 10 oncocytic adenomas, 10 follicular adenomas (FA), 10 FTC, 6 ATC, 12 PTC using two microsatellite markers for HGF, and two for c-met. LOH for all 4 markers was found in 100% of FTC, 100% of ATC, and (for only 1 or 2 markers) in 10-29% of FA. This is the first demonstration of an LOH that separates both FTC and ATC from PTC, in the best possible manner: 100% vs. 0%. Clearly, each of the two segments we have probed contains at least one tumor suppressor gene, whose inactivation is crucial for the establishment of the FTC (and ATC) phenotype. This loss of genetic material explains why FTC and ATC, but not PTC, fail to express both HGF and c-met. Our findings may also have immediate diagnostic application, in the context of assisting pathologists in the often difficult task of distinguishing FA from FTC.

DNA extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction.

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.

How healthcare professionals contribute to hope in patients with cancer.

PURPOSE/OBJECTIVES: To explore whether healthcare professionals influence the level of hope in patients with cancer and, if so, how they influence their hope. DESIGN: Descriptive, qualitative design. SETTING: An adult hematology/oncology unit in the upper midwestern United States. SAMPLE: Thirty-two men and women receiving active or supportive treatment or palliative care for cancer. METHODS: Semistructured interviews conducted in the participants' hospital rooms. Ten investigators and two consultants transcribed and analyzed the interview data using content analysis. They identified themes and subthemes that described healthcare professionals' roles. MAIN RESEARCH VARIABLES: Healthcare professionals' contributions to hope as described by patients with cancer. FINDINGS: Healthcare professionals positively and negatively influenced hope in this sample. Hope was facilitated by being present, giving information, and demonstrating caring behaviors. Negative influences on hope primarily concerned the way in which healthcare professionals gave information. CONCLUSION: Healthcare professionals do influence patients' perceptions of their hope. Although most nursing actions are hope enhancing, nurses can reduce a patient's sense of hope if information provided or attitude toward the patient is insensitive or disrespectful. IMPLICATIONS FOR NURSING PRACTICE: Nurses can increase patients' hope by being present, taking time to talk, and being helpful. They must provide information and answer questions in a compassionate, positive, honest, and respectful manner. Caring behaviors such as thoughtful gestures, showing warmth and genuineness, and being friendly and polite also increase patients' hope.

Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.

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.

DNAzol: a reagent for the rapid isolation of genomic DNA.

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.

Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources.

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.

One-hour downward capillary blotting of RNA at neutral pH.

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.

Endothelial modulation of renal interlobar arteries from pregnant rats.

The purpose of this study was to determine whether there are gestational effects on 1) the response of resistance arteries from the renal vasculature to phenylephrine and 2) the endothelial modulation of these arteries. Interlobar arteries (200-300 microns ID) were isolated from the kidneys of virgin and pregnant rats at 18-20 days of gestation (term, 22 +/- 1 days) and studied in a pressurized arteriograph system. Intact arteries from virgin and pregnant rats did not differ in sensitivity to phenylephrine. Arteries without endothelium from both groups were more sensitive to phenylephrine than arteries with endothelium. Sensitivity was increased 3.4-fold by endothelial removal in arteries from virgin rats and 1.5-fold in the pregnant group. Concentration-response relationships to phenylephrine were determined in arteries with endothelium and then repeated in the presence of 2.5 x 10(-4) M N omega-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase. All arteries were more sensitive to phenylephrine in the presence of L-NNA, with an average increase of 3.2-fold for the arteries from virgin rats and 1.6-fold from pregnant rats. These results indicate that the increased sensitivity to phenylephrine is primarily due to elimination of endothelium-derived relaxing factor (EDRF) and that basal EDRF activity is decreased during late gestation. To determine whether stimulated endothelium-dependent relaxation is enhanced in pregnancy, arteries with endothelium were constricted with phenylephrine to 50% of their maximum and relaxed to increasing concentrations of methacholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Composition and mechanics of mesenteric resistance arteries from pregnant rats.

We tested the hypothesis that the systemic resistance vasculature of the rat is remodeled during pregnancy as evidenced by significant alterations in the passive mechanical properties and extracellular matrix proteins in mesenteric arteries. Mechanical characteristics were determined for arteries from 20-day pregnant rats (n = 6) and age-matched controls (n = 5). Lumen diameter and wall thickness were measured in pressurized arteries (250-microns diameter) using a dimension analyzing system. Distensibility (the relative change in diameter per unit change in pressure) was less in the arteries from the pregnant rats (P less than 0.01). The calculated stress-strain relationships and elastic moduli indicated that the arteries were less stiff by late gestation (P less than 0.05). Ultramicro amino acid analysis and radioimmunoassay were used to measure hydroxyproline, desmosine, and leucine as indicators of collagen, elastin, and total protein, respectively, in similar-sized arteries. Hydroxyproline/leucine (index of collagen) and desmosine/leucine (elastin concentration) decreased 19 and 15% by late gestation (P less than 0.05). The significant alterations in passive mechanics and in extracellular protein content support the concept that arterial wall remodeling in the peripheral vasculature may be one component of the cardiovascular adaptations during pregnancy.

An outbreak of hepatitis B associated with jet injections in a weight reduction clinic.

From January 1984 through November 1985, 31 clinical cases of hepatitis B occurred among attendees of a weight reduction clinic (clinic 1). Before the onset of illness, each case-patient had received a series of injections of human chorionic gonadotropin administered by jet injectors at clinic 1. Clinical history, risk factor assessment, serologic evaluation, and review of clinic injection records were obtained on 287 (84%) of 341 persons who had attended clinic 1 in the first 6 months of 1985. Of this cohort, 21% (60/287) had evidence of acute infection with hepatitis B virus (either documented clinical cases or antibody to hepatitis B core antigen, IgM positive). Of persons who had been given human chorionic gonadotropin at the clinic during the period studied, 24% (57/239) of those receiving human chorionic gonadotropin only by jet injector experienced acute hepatitis B virus infection. None of the 22 persons who had received injections only by syringe experienced hepatitis B virus infection. Stopping the use of the jet injectors on July 2, 1985, at clinic 1, was associated with the termination of this outbreak. This investigation demonstrated that jet injectors can become contaminated with hepatitis B virus and then may be vehicles for its transmission.

Defining the differences between crisis intervention and short-term therapy.

Using the model of "person plus stress yields reaction," the authors discuss the differences between crisis intervention and short-term treatment, including psychiatric emergencies. In emergency treatment the central focus is on the reaction, or symptoms, while in crisis intervention the emphasis is on the stress and its quick resolution. In short-term treatment the focus is on the person and exploration of behavior patterns and feelings. The authors believe that the number of crisis cases handled by a therapist must be limited because of their exhausting nature.