A simple method for the quantitative isolation of undegraded high molecular weight ribonucleic acid

Barlow, J. J.; Mathias, A. P.; Williamson, Russell; Gammack, D. B.

doi:10.1016/0006-291x(63)90163-3

Cited by 142 publications

(56 citation statements)

References 3 publications

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“…For RNA gel blot analysis, total RNA was isolated from various tissues using lithium chloride buffer [100 mm lithium chloride, 100 mm Tris HCl (p.H. 8.0), 10 mm EDTA, 1% SDS] and precipitated with 4 m lithium chloride (Barlow et al 1963). Total RNA was fractionated on denaturing gels, blotted onto nylon membranes, and hybridized with a 32 P-dCTP probe fragments as described previously (Boddu et al 2004).…”

Section: Methodsmentioning

confidence: 99%

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

Sekhon¹,

Chopra

2009

Genetics

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Maize pericarp color1 (p1) gene, which regulates phlobaphene biosynthesis in kernel pericarp and cob glumes, offers an excellent genetic system to study tissue-specific gene regulation. A multicopy p1 allele, P1-wr (white pericarp/red cob) is epigenetically regulated. Hypomethylation of P1-wr in the presence of Unstable factor for orange1 (Ufo1), leads to ectopic pigmentation of pericarp and other organs. The Ufo1-induced phenotypes show incomplete penetrance and poor expressivity: gain of pigmentation is observed only in a subset of plants carrying Ufo1 mutation, and the extent of pigmentation is highly variable. We show that Ufo1 induces progressive hypomethylation of P1-wr repeats over generations. After five generations of exposure to Ufo1, a 30-40% decrease in CG and CNG methylation was observed in an upstream enhancer and an intron region of P1-wr. Interestingly, such hypomethylation correlated with an increase in penetrance of the Ufo1-induced pigmentation phenotype from $27 to 61%. Expressivity of the Ufo1-induced phenotype also improved markedly as indicated by increased uniformity of pericarp pigmentation in the later generations. Furthermore, the poor expressivity of the Uo1 is associated with mosaic methylation patterns of the P1-wr upstream enhancer in individual cells and distinct P1-wr gene copies. Finally, comparison of methylation among different tissues indicated that Ufo1 induces rapid CG and CNG hypomethylation of P1-wr repeats during plant development. Together, these data indicate that the poor penetrance and expressivity of Ufo1-induced phenotypes is caused by mosaicism of methylation, and progressive mitotic hypomethylation leads to improved meiotic heritability of the mutant phenotype. In duplicated genomes like maize, loss of an epigenetic regulator may produce mosaic patterns due to redundancy of epigenetic regulators and their target sequences. We show here that multiple developmental cycles may be required for complete disruption of suppressed epigenetic states and appearance of heritable phenotypes.

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Section: Methodsmentioning

confidence: 99%

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

Sekhon¹,

Chopra

2009

Genetics

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“…Total RNA was extracted from liver samples using TRI Reagant isolation solution with sequential high salt (Molecular Research Center) and LiCl precipitation steps intended to remove glycogen contamination (Barlow et al 1963). The mean 260:230 nm ratio for these RNA samples ranged from 1 .…”

Section: Rna Isolation For Quantitative Real-time Pcrmentioning

confidence: 99%

Regulation of endocrine and paracrine sources of Igfs and Gh receptor during compensatory growth in hybrid striped bass (Morone chrysops×Morone saxatilis)

Picha

Turano

Tipsmark

et al. 2008

Journal of Endocrinology

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Compensatory growth (CG) is a period of growth acceleration that exceeds normal rates after animals are alleviated of certain growth-stunting conditions. In hybrid striped bass (HSB, Morone chrysops!Morone saxatilis), 3 weeks of complete feed restriction results in a catabolic state that, when relieved, renders a subsequent phase of CG. The catabolic state was characterized by depressed levels of hepatic Type I and II GH receptor (ghr1, ghr2) and igf1 mRNA, along with considerable decreases in plasma Igf1. The state of catabolism also resulted in significant declines in hepatic igf2 mRNA and in circulating 40 kDa Igfbinding protein (Igfbp). Skeletal muscle expression of ghr2 mRNA was significantly increased. Upon realimentation, specific growth rates (SGRs) were significantly higher than sized-matched controls, indicating a period of CG. Hepatic ghr1, ghr2, igf1 and igf2 mRNA levels along with plasma Igf1 and 40 kDa Igfbp increased rapidly during realimentation. Plasma Igf1 and total hepatic igf2 mRNAwere significantly correlated to SGR throughout the study. Skeletal muscle igf1 mRNA also increased tenfold during CG. These data suggest that endocrine and paracrine/autocrine components of the GH-Igf axis, namely igf1, igf2, and ghr1 and ghr2, may be involved in CG responses in HSB, with several of the gene expression variables exceeding normal levels during CG. We also demonstrate that normalization of hepatic mRNA as a function of total liver production, rather than as a fraction of total RNA, may be a more biologically appropriate method of quantifying hepatic gene expression when using real-time PCR.

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“…Northern blot analysis Total cellular RNA was isolated by the ureum/lithium chloride method (Barlow et al, 1963) from cell lines which express high levels (HBL-100 and T47D), low levels (OVCAR-4 and A375) and no (K562) 34 protein.…”

Section: Materials and Methods Cdna Cloningmentioning

confidence: 99%

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain.

et al. 1990

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A simple method for the quantitative isolation of undegraded high molecular weight ribonucleic acid

Cited by 142 publications

References 3 publications

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

Regulation of endocrine and paracrine sources of Igfs and Gh receptor during compensatory growth in hybrid striped bass (Morone chrysops×Morone saxatilis)

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain.

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