Isolation, Purification, and Fractionation of RNA

Poulson, Rozanne

doi:10.1007/978-1-4612-6360-9_10

Cited by 16 publications

(2 citation statements)

References 222 publications

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“…Materials and their sources were as follows: guanidinium thiocyanate (GSCN), Tridom Chemical (Hauppauge, NY); cesium chloride, Bethesda Research Laboratories (Gaithersburg, MD); bentonite from Sigma (St. Louis, MO), further purified by the method of Poulson (1973); vanadyl sulfate (Fisher, Medford, MA); sucrose, density gradient grade, Schwarz-Mann (Spring Valley, NY); oligo d(T)-cellulose, Type T3, Collaborative Research (Waltham, MA); [3H]ribosomal RNA, Miles (Elkhart, IN); ~-[~~S]methionine (> 1000 Ci/mmol, translation grade), rabbit reticulocyte lysate protein synthesis components, adenovirus-2 mRNA, globin mRNA, and Gene Screen Hybridization Transfer Membranes (HTM), New England Nuclear (Boston, MA); molecular weight marker proteins, Biorad (Rockville Centre, NY); nitrocellulose membranes (HAHY304FO), Millipore (Bedford, MA). All reagents used for the preparation and processing of polyacrylamide gels were listed in previous reports (Marotta et al, 1978a(Marotta et al, , 1981a.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Sajdel-Sulkowska

Coughlin

Marotta

1983

Journal of Neurochemistry

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Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)‐containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)‐cellulose‐purified poly(A)‐containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L‐[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9‐ to 20‐fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two‐dimensional polyacrylamide gel electrophoresis (PAGE): size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)‐cellulose chromatography yielded 1.8 μg/g and 2.0 μg/g, respectively, of poly(A)‐containing mRNA; this represents a two‐ to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two‐dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.

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Section: Methodsmentioning

confidence: 99%

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Sajdel-Sulkowska

Coughlin

Marotta

1983

Journal of Neurochemistry

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“…These compounds purportedly function by eliminating all intrinsic enzymatic activity and have been incorporated into RNA isolation procedures with varying degrees of success. The preparation of these clays for the elimination of RNase activity has been described elsewhere (Fraenkel-Conrat, et al, 1961;Poulson, 1973;Blumberg, 1987) and not included here, as they are no longer widely used to control RNase. Many of these are older and no longer in widespread use, though their persistence in the literature mandates mentioning here.…”

Section: Non-specific Inhibitorsmentioning

confidence: 99%

Resilient Ribonucleases

Farrell

2010

RNA Methodologies

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Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes

2001

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Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2-4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68 degrees C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

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Isolation, Purification, and Fractionation of RNA

Cited by 16 publications

References 222 publications

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Resilient Ribonucleases

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes

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