Determining suitable internal standards for mRNA quantification of increasing cancer progression in human breast cells by real-time reverse transcriptase polymerase chain reaction

Morse, David L.; Carroll, Danielle; Weberg, Lyndon; Borgstrom, Mark; Ranger‐Moore, James; Gillies, Robert J.

doi:10.1016/j.ab.2005.03.034

Cited by 64 publications

(61 citation statements)

References 26 publications

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“…These results seem to underline the general supposition that 18S expression can be relatively stable and had in fact been suggested for normalization in other cell types and conditions. 9,13,14 Fluctuation of rRNA levels especially of 28S are nonetheless apparent in exfoliated cells, influence total RNA levels accordingly, and might therefore not adequately represent mRNA activities. The finding that densitometric estimation of total RNA mass by the intensity of rRNA bands was completely independent from all PCR quantifications-DNA and RNA-measured in the sample could originate from different degrees of partial degradation that influences UV absorbance of ribosomal bands but not the abundance of short PCR-amplified target templates.…”

Section: Discussionmentioning

confidence: 99%

“…A master mix of primers and exogenous plant gene spike chlorophyll A-B binding protein (CAB) (Stratagene, La Jolla, CA) was prepared. Aliquots were added to each sample (final concentrations per sample were 300 ng of random primer, 50 ng of oligo-T [12][13][14][15][16][17][18] [Invitrogen], and 0.1 pg of CAB mRNA), and the mixtures were heated at 65°C for 5 minutes and then transferred to ice. After 1 minute on ice, the reaction mix was added (2 l of RT buffer, 2 l of dithiothreitol [100 mmol/L], 2 l of dNTPs [10 mmol/L], 1 l of dH 2 O, and 1 l of Superscript III reverse transcriptase [Invitrogen]) to the final reaction volume of 20 l. Samples were incubated at 25°C for 5 minutes, 50°C for 50 minutes, and 70°C for 15 minutes.…”

Section: Cdna Synthesismentioning

confidence: 99%

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DNA and RNA References for qRT-PCR Assays in Exfoliated Cervical Cells

Steinau

Rajeevan

Unger

2006

The Journal of Molecular Diagnostics

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The noncritical use of housekeeping genes, RNA mass, or cell number for normalization in quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays has come under scrutiny in recent years, highlighting the need to evaluate references in the immediate context of the relevant samples and experimental design. The purpose of this study was to select appropriate references for normalizing qRT-PCR assays of gene expression in exfoliated cervical cells. We used total nucleic acid extracts from 30 samples, representing the full spectrum of pre-invasive cervical neoplasia. We determined the DNA content by quantitative PCR for the single-copy gene ␤-globin and total RNA content using quantitative image analysis of ribosomal bands. In addition, qRT-PCR for 13 candidate housekeeping genes was performed. We used two analysis methods, geNorm and NormFinder, to identify the best combination of reference genes and then correlated housekeeping gene expression with DNA content and gel representation of ribosomal RNA. ACTB was the most stable single gene. The addition of PGK1 and RPLP0 increased the robustness in qRT-PCR applications not stratified by disease. These genes also showed the highest correlation with DNA contents in the same samples. If special attention to intraepithelial lesions is appropriate , RPL4 and PGK1 are recommended as the best combination of two genes.

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Section: Discussionmentioning

confidence: 99%

Section: Cdna Synthesismentioning

confidence: 99%

DNA and RNA References for qRT-PCR Assays in Exfoliated Cervical Cells

Steinau

Rajeevan

Unger

2006

The Journal of Molecular Diagnostics

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“…Relative expression values were normalized to a standard curve using cDNA prepared from a 5-week-old CD1 mouse mammary gland and also to 18S expression levels for each individual cDNA as previously described (Nicolas et al, 2003;Zhou et al, 2003;Morse et al, 2005). Additional information on quality control and 18S normalization can be found in Supplementary data 4.…”

Section: Rt-pcr and Western Analysismentioning

confidence: 99%

CITED1 homozygous null mice display aberrant pubertal mammary ductal morphogenesis

et al. 2005

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“…The expression of ␤-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), EPO, and SDF-1 was also evaluated by real-time PCR as reported elsewhere. 17,22,23 Human kidney RNA (BD Biosciences Clontech) served as a positive control for EPO expression. …”

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confidence: 99%

Von Hippel-Lindau–dependent polycythemia is endemic on the island of Ischia: identification of a novel cluster

Perrotta¹,

Nobili²,

Ferraro³

et al. 2006

Blood

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Chuvash polycythemia (MIM 263400) is an autosomal recessive disorder characterized by a high hemoglobin level, relatively high serum erythropoietin, and early death. It results from a Von Hippel-Lindau (VHL) gene mutation (C598T) that causes increased HIF-1␣ activity and erythrocyte production in the face of normoxia. This polycythemia is endemic in Chuvashia, whereas its worldwide frequency is very low. We investigated the incidence of the Chuvash-type VHL mutation in Campania (South Italy) and identified 14 affected subjects (5 families). Twelve live on the island of Ischia (Bay of Naples). From analysis of the mutated allele, we found that the disease was more frequent on Ischia (0.070) than in Chuvashia (0.057). The haplotype of all patients matched that identified in the Chuvash cluster, thereby supporting the single-founder hypothesis. We also found that nonaffected heterozygotes had increased HIF-1␣ activity, which might confer a biochemical advantage for mutation maintenance. In conclusion, we have identified the first large cluster of Chuvash erythrocytosis outside Chuvashia, which suggests that this familial polycythemia might be endemic in other regions of the world. IntroductionThe Chuvash variant of familial polycythemia was first described in more than 100 individuals from about 80 families living in the mid-Volga River region of European Russia. 1 The disease is characterized by a high hemoglobin level, increased plasma erythropoietin (Epo) level, varicose veins, vertebral hemangiomas, low blood pressure, and an elevated serum concentration of vascular endothelial growth factor (VEGF). 2 Patients affected by Chuvash polycythemia die early, mainly as a result of cerebral vascular events or peripheral thrombosis. These injuries seem to be linked to mechanisms other than blood hyperviscosity or serum Epo content. 2 Indeed, the prevalence of low blood pressure in patients with Chuvash polycythemia contrasts with the hypertension frequently associated with polycythemia vera and other familial polycythemias resulting from excess Epo.Genome-wide screening and candidate gene characterization demonstrated that the Arg200Trp mutation (C598T) of the Von Hippel-Lindau (VHL) gene causes the Chuvash form of polycythemia. 3 Thereafter, the mutation was detected in homozygosity in patients with sporadic or familial congenital erythrocytosis from diverse ethnic groups. 4-8 However, 19 homozygotes have been identified among the more than 150 known cases of non-Chuvash familial erythrocytosis. 9 Furthermore, 8 other VHL mutations (Arg79Cys, Gly104Val, Asp126Tyr, Val130Leu, Gly144Arg, Tyr175Cys, Leu188Val, His191Asp, Pro192Ala) were detected in either homozygotes or compound heterozygotes. 4,5,7,8,10 These mutations were detected in a total of 10 cases, which indicates that the C598T transition is the major cause of VHL-related erythrocytosis.The C598T mutation likely originated from a single founder event because the VHL haplotype in non-Chuvash patients is identical to that in polycythemic patients from Chuvashia....

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Determining suitable internal standards for mRNA quantification of increasing cancer progression in human breast cells by real-time reverse transcriptase polymerase chain reaction

Cited by 64 publications

References 26 publications

DNA and RNA References for qRT-PCR Assays in Exfoliated Cervical Cells

DNA and RNA References for qRT-PCR Assays in Exfoliated Cervical Cells

CITED1 homozygous null mice display aberrant pubertal mammary ductal morphogenesis

Von Hippel-Lindau–dependent polycythemia is endemic on the island of Ischia: identification of a novel cluster

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