Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements

Walker, Jerilyn A.; Hughes, David A.; Hedges, Dale J.; Anders, Bridget A; Laborde, Meredith E.; Shewale, Jaiprakash G.; Sinha, Sudhir K.; Batzer, Mark A.

doi:10.1016/j.ygeno.2003.09.003

Cited by 59 publications

(43 citation statements)

References 29 publications

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“…At death, lungs were dissected for DNA extraction. The assay for rat DNA was adapted from the method described by Walker et al (36). Bioluminescent reporter imaging was performed to monitor the lung seeding of ELT3-Luciferase cells.…”

Section: Methodsmentioning

confidence: 99%

Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells

Robb

Morrison

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

157

198

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Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-␤-estradiol (E2) causes a 3-to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E 2-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E 2 inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E 2 enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEKdependent pathways by E 2 leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.L AM, the pulmonary manifestation of tuberous sclerosis complex (TSC), affects women almost exclusively (1). LAM affects 30Ϫ40% of women with TSC (2, 3). In a Mayo Clinic series, LAM was the third most frequent cause of TSC-related death, after renal disease and brain tumors (4). LAM can also occur in women who do not have germline mutations in TSC1 or TSC2 (sporadic LAM). LAM cells from both TSC-LAM and sporadic LAM carry inactivating mutations in both alleles of the TSC1 or TSC2 genes (5). The protein products of TSC1 and TSC2, hamartin and tuberin, respectively, form heterodimers (6, 7) that inhibit the small GTPase Ras homologue enriched in brain (Rheb), via tuberin's highly conserved GTPase activating domain. In its active form, Rheb activates the mammalian target of rapamycin (mTOR) complex 1 (TORC1), which is a key regulator of protein translation, cell size, and cell proliferation (8). Evidence of TORC1 activation, including hyperphosphorylation of ribosomal protein S6, has been observed in tumor specimens from TSC patients and LAM patients (9-11). Independent of its activation of mTOR, Rheb inhibits the activity of B-Raf and C-Raf/Raf-1 kinase, resulting in reduced phosphorylation of p42/44 MAPK (12-14), but the impact of the Raf/MEK/ MAPK pathway on disease pathogenesis is undefined.LAM is characterized pathologically by widespread proliferation of abnormal smooth muscle cells and by cystic changes within the lung parenchyma (1). About 60% of women with the sporadic form of LAM also have renal angiomyolipomas. The presence of TSC2 mutations in LAM cells and renal angiomyolipoma cells from women with sporadic LAM, but not in normal tissues, has led to the hypothesis that LAM cells spread to the lungs via a metastatic mechanism, despite the fact that LAM cells have a histologically benign appearance (15,16). Genetic and fluorescent in situ hybridization analyses of recurrent LAM after lung transplantation support this benign metastatic model (16).The female...

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Section: Methodsmentioning

confidence: 99%

Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells

Robb

Morrison

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

157

198

View full text Add to dashboard Cite

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“…1). The PCR-amplified products are specific for each host species, or family, as in the case of pigs 19,20 20 . Next, a denaturation step at 95 ºC for five minutes followed by 35 cycles 95 ºC for one minute, 58 ºC for one minute and 72 ºC for one minute, with a final extension at 72 ºC for seven minutes (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Temporal differences in blood meal detection from the midguts of Triatoma infestans

Pinto

Roellig

Gilman

et al. 2012

Rev. Inst. Med. trop. S. Paulo

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SUMMARYWe used genus/species specific PCRs to determine the temporal persistence of host DNA in Triatoma infestans experimentally fed on blood from six common vertebrate species: humans, domestic dogs, guinea pigs, chickens, mice, and pigs. Twenty third or fourth instar nymphs per animal group were allowed to feed to engorgement, followed by fasting-maintenance in the insectary. At 7, 14, 21, or 28 days post-feeding, the midgut contents from five triatomines per group were tested with the respective PCR assay. DNA from all vertebrate species was detected in at least four of five study nymphs at seven and 14 days post-feeding. DNA of humans, domestic dogs, guinea pigs, pigs, and chickens were more successfully detected (80-100%) through day 21, and less successfully (20-100%) at day 28. Findings demonstrate that species-specific PCRs can consistently identify feeding sources of T. infestans within two weeks, a biologically relevant time interval.

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“…Con el fin de comprobar la presencia de ADN amplificable (buena calidad) y ausencia de inhibidores luego de la extracción a partir de las muestras se desarrolló una reacción de amplificación utilizando iniciadores dirigidos a material genético que debería estar presente en los mismos. Dichos iniciadores (Walker et al 2004) son específicos para una porción no codificante y conservada de genoma de rata, ratón y hámster y generan amplificaciones de 118 pb. Este control es de gran importancia en los casos en los que las reacciones de amplificación para Leishmania dieron no detectable.…”

Section: Biología Molecularunclassified