Isolation RNA

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

The liver RNA isolated from rats treated with C-NHEX yielded 1,6-hexanediol upon hydrolysis with acid (54). This result indicates that NHEX underwent metabolic a-hydroxylation to give an adduct that may have been formed at 0 of guanine. The initially formed adduct would have been expected to be a 6-oxo-hexyl derivative reduction of the adduct must have occurred in order to produce the observed 1,6-hexanediol. 

The second experiment was carried out with the same RNA isolated from mycelia growing on presence of glucose and apple pectin. cDNAs were obtained from those RNA by reverse trancription and used as template for PCR assays. Specific oligonucleotide primers for PG gene were used in the amplification reaction. The Fig-5 shows the results of this amplification experiment. 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

Fig. 14.5. Composite of Northern blots prepared with RNA isolated from different A. suum larval stages and adult tissues and probed with A. suum cDNAs specific for E1al (Johnson etal., 1992) E1all (Johnson etal., 1992) ...

There are numerous protocols for polysomal gradients preparations that differ mainly at the step for harvesting the cells, and the gradient composition and separation times. The protocol presented later was optimized for isolation of polysomal mRNA from the yeast Saccharomyces cerevisiae, yet many steps will be similar to other eukaryotes and the procedure can easily be modified for other organisms. We will use this protocol as a template on which we will indicate and highlight points that are critical for the microarray analysis. Generally, the RNA isolated by this protocol can be used for analysis by DNA microarray, Northern blot, or RT-PCR. 

Macabeo- Oral tissues of RNA Isolation kit Real time RNA can be... 

Abrahamsen Lymph nodes RNA isolation kit Real-time, for All three markers... 

Following a procedure modified from the protocol of Newman [Newman et al. 1990], total RNA was extracted from 100 ml C. moewusii culture after 2 hours of anaerobic adaptation. The mRNA was isolated from 250 pg of total RNA using the Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). 50 pg RNA isolated from anaerobically induced cultures of C. moewusii (1 h, 2 h, 3 h and 4 h) were separated on a 0.8 % agarose gel... 

A Anaerobic adaptation was obtained by flushing the cells with argon. At indicated time points, samples were taken to measure the in vitro Fk-ase activity of C. reinhardtii (box), S. obliquus (circle), C. fusca (triangle), C. moewusii (diamond). While the activities of both Scenedesmus species are comparative low, the in vitro H -production rate of anaerobically induced C. moewusii cultures is 2 times higher than the activity of induced C. reinhardtii cultures. B Northern blots with equal amounts of total RNA isolated from an anaerobically adapted culture (2 h) and an uninduced reference culture (0 h) of C. moewusii. The upper blot was incubated with a RNA sample of the 3 UTR of the hydA 1 cDNA, while the lower blot was incubated with a RNA sample generated of the cDNA from the constitutively expressed sedoheptulose 1,7-bisphosphatasegene. 

Messenger RNA isolated from differentiated HL-60 cells is thus a convenient source of transcripts for the receptor, and this mRNA (50-100 ng) can be injected into X. laevis oocytes. These oocytes actively translate foreign mRNA molecules, and often the expressed protein is functional 2-5 days later, functional fMet-Leu-Phe receptors can be detected on the cell surface. The identity of a functional fMet-Leu-Phe receptor was confirmed because ... 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

Chenchik, A., Moqadam, F. and Siebert, P. A new method for full-length cDNA cloning by PCR. In A Laboratory Guide to RNA Isolation, Analysis and Synthesis. PA. Krieg, (ed.) Wiley Liss, Inc. pp. 273-321, 1996. 

RNA Isolation from Laser-Capture Microdissected Samples. 355... 

During recent years, many of our appheations of competitive RT-PCR have involved the analysis of RNA from very small tissue samples obtained by laser capture microdissection. We have therefore adopted a modified reamphfication protocol (competitive Nested-RT-PCR) for the quantitation of very small tissue samples (Costa et al., 2002). The procedure involves the use of very low concentrations of cRNA (internal standard) in the presence of total RNA isolated from a measured and microdissected tissue sample or a... 

The first step in all RNA isolation protocols involves lysing the cell in a chemical environment that denatures ribonucleases. The RNA is then fractionated from other cellular macromolecules by either homogenizing the tissue (dissected brain tissue) or simply vortexing the sample (very small tissues and laser-microdissected sample) without further homogenization. The cell type from which the RNA is to be isolated, the sample size, and the eventual use of the RNA will determine which procedure described here is appropriate. 

The routine competitive RT-PCR protocol described earlier is modified for the quantitative nested RT-PCR analysis of RNA isolated from laser-microdissected samples. The low amount of starting template and the... 

Differential display analysis of hippocampal RNA Isolated from Individual out-bred Long Evans Hooded (LEH), seizure-prone (SP), and seizure-resistant (SR) rats... 

The following materials are provided with the ToTALLY RNA RNA isolation kit (Ambion, Austin, Tx) ... 

The lymphocytes are centrifuged at 270g for lOmin at 4°C. The supernatant is removed by aspiration and stored at 4°C (see Note 16). The lymphocytes in the pellet are washed once with ice-cold PBS and recentrifuged at 270g for lOmin at 4°C (see Note 14). The pellet containing the restimulated lymphocytes is used for total RNA isolation. 

After in vitro restimulation with the antigen, the cells are collected by centrifugation, washed in cold PBS, and used for RNA isolation (see Note 3). The methods... 

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