Sample Preparation From Blood, Cells, and Other Fluids

Kawasaki, Ernest S.

doi:10.1016/b978-0-12-372180-8.50022-6

Cited by 316 publications

(213 citation statements)

References 4 publications

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“…Genomic DNA was isolated from liver biopsy specimens at the same time as RNA by standard methods. 13 DNA bisulfite modification was performed as descibed. 14 Briefly, this technique was based on bisulfite treatment of genomic DNA, which converts all the unmethylated cytosines to uracils while conserving the methylated cytosines; we used the EZ DNA Methylation Kit and followed the manufacturer's protocol (Zymo Research Corporation, Orange, CA).…”

Section: Methodsmentioning

confidence: 99%

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

et al. 2010

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Insulin resistance (IR) and mitochondrial dysfunction play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic factors and epigenetic modifications occurring in the liver contribute to the IR phenotype. We specifically examined whether fatty liver and IR are modified by hepatic DNA methylation of the peroxisome proliferator-activated receptor c coactivator 1a (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters, and also evaluated whether liver mitochondrial DNA (mtDNA) content is associated with NAFLD and IR. We studied liver biopsies obtained from NAFLD patients in a case-control design. After bisulfite treatment of DNA, we used methylation-specific polymerase chain reaction (PCR) to assess the putative methylation of three CpG in the PPARGC1A and TFAM promoters. Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was evaluated by way of realtime PCR. Liver PPARGC1A methylated DNA/unmethylated DNA ratio correlated with plasma fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA-IR); TFAM methylated DNA/unmethylated DNA ratio was inversely correlated with insulin levels. PPARGC1A promoter methylation was inversely correlated with the abundance of liver PPARGC1A messenger RNA. The liver mtDNA/nDNA ratio was significantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA-IR, fasting glucose, and insulin and was inversely correlated with PPARGC1A promoter methylation. Conclusion: Our data suggest that the IR phenotype and the liver transcriptional activity of PPARGC1A show a tight interaction, probably through epigenetic modifications. Decreased liver mtDNA content concomitantly contributes to peripheral IR.

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Section: Methodsmentioning

confidence: 99%

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

et al. 2010

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“…DNA was extracted by phenol-chloroform and precipitated with ethanol. Eight samples with small numbers of cells were prepared by resuspension in digestion mixture (1 x PcR buffer, 0.25% Tween 20, 0.6 yl of 10 mg ml-' proteinase K per 100 ;LI) at a concentration of 5 x I05 cell ml-' and incubated at 56°C for 2'h followed by a 95°C 20 min inactivation step to inhibit proteinase K (Kawasaki, 1990).…”

mentioning

confidence: 99%

Detection of bcl-2/JH rearrangement in follicular and diffuse lymphoma: concordant results of peripheral blood and bone marrow analysis at diagnosis

Yuan

Dowling²,

Zucca³

et al. 1993

Br J Cancer

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“…PCR for GBE amplification of genomic DNA samples isolated from blood [18] was done in separate reactions using a common forward primer and either of 2 allele-specific (normal or deleted) reverse primers flanking the site of the 5' breakpoint. PCR primers were 5'-TGTATCTTCCACATCAGAGCAGTG-3' (GSD F), 5'-ATAGTGTAGGTGTTGCATCCCTAAT-3' (normal allele R), and 5'-GACACACCTGTAATCAAGTACCTCT-3' (deleted allele R).…”

Section: Mutation Detection and Carrier Testingmentioning

confidence: 99%

A complex rearrangement in GBE1 causes both perinatal hypoglycemic collapse and late-juvenile-onset neuromuscular degeneration in glycogen storage disease type IV of Norwegian forest cats

Fyfe

Kurzhals

Hawkins

et al. 2007

Molecular Genetics and Metabolism

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Deficiency of glycogen branching enzyme (GBE) activity causes glycogen storage disease type IV (GSD IV), an autosomal recessive error of metabolism. Abnormal glycogen accumulates in myocytes, hepatocytes, and neurons, causing variably progressive, benign to lethal organ dysfunctions. A naturally occurring orthologue of human GSD IV was described previously in Norwegian forest cats (NFC). Here we report that while most affected kittens die at or soon after birth, presumably due to hypoglycemia, survivors of the perinatal period appear clinically normal until onset of progressive neuromuscular degeneration at 5 months of age. Molecular investigation of affected cats revealed abnormally spliced GBE1 mRNA products and lack of GBE cross-reactive material in liver and muscle. Affected cats are homozygous for a complex rearrangement of genomic DNA in GBE1, constituted by a 334 bp insertion at the site of a 6.2 kb deletion that extends from intron 11 to intron 12 (g. IVS11+1552_IVS12-1339 del6.2 kb ins334 bp), removing exon 12. An allele-specific, PCR-based test demonstrates that the rearrangement segregates with the disease in the GSD IV kindred and is not found in unrelated normal cats. Screening of 402 privately owned NFC revealed 58 carriers and 4 affected cats. The molecular characterization of feline GSD IV will enhance further studies of GSD IV pathophysiology and development of novel therapies in this unique animal model.

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Sample Preparation From Blood, Cells, and Other Fluids

Cited by 316 publications

References 4 publications

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

Detection of bcl-2/JH rearrangement in follicular and diffuse lymphoma: concordant results of peripheral blood and bone marrow analysis at diagnosis

A complex rearrangement in GBE1 causes both perinatal hypoglycemic collapse and late-juvenile-onset neuromuscular degeneration in glycogen storage disease type IV of Norwegian forest cats

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