A Method to Monitor mRNA Levels in Human Breast Tumor Cells Obtained by Fine-needle Aspiration

Lange, Majella S. de; Top, B.; Lambrechts, Caro; Maas, Riks; Peterse, Hans; Mooi, Wolter J.; Veer, Laura J. van’t; Rodenhuis, S.

doi:10.1097/00019606-199712000-00008

Cited by 7 publications

(3 citation statements)

References 14 publications

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“…In contrast, previous studies have pointed to advantages of PBGD as an internal standard (Lin et al ., 1991; de Lange et al ., 1997; Fink et al ., 1999).…”

Section: Participants and Methodsmentioning

confidence: 99%

Quantification of telomerase activity, porphobilinogen deaminase and human telomerase reverse transcriptase mRNA in testicular tissue – new parameters for a molecular diagnostic classification of spermatogenesis disorders

et al. 2002

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The aim of the study was to evaluate the quantitative detection of human telomerase reverse transcriptase (hTERT) mRNA and telomerase activity as new molecular diagnostic parameters for a subclassification of spermatogenesis disorders. Telomerase activity was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by fluorescence real-time RT-PCR in a LightCycler in cryopreserved testicular tissue specimens. This was paralleled by a histological workup. The discriminant analysis showed that detection of normalized hTERT expression was able to correctly classify 89.0% of the investigated tissue specimens into the subgroups of full spermatogenesis, maturation arrest or Sertoli-cell-only syndrome. In contrast, discriminant analysis revealed an only 58% accuracy of telomerase activity for the investigated tissue specimens. This study shows that the quantification of hTERT expression in testicular tissue by real-time fluorescence RT-PCR is well suited for correctly classifying spermatogenesis disorders and proved to be markedly superior to the determination of telomerase activity.

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“…In contrast, previous studies have pointed to advantages of PBGD as an internal standard (Lin et al ., 1991; de Lange et al ., 1997; Fink et al ., 1999).…”

Section: Participants and Methodsmentioning

confidence: 99%

Quantification of telomerase activity, porphobilinogen deaminase and human telomerase reverse transcriptase mRNA in testicular tissue – new parameters for a molecular diagnostic classification of spermatogenesis disorders

et al. 2002

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“…For the estimation of VEGF and other angiogenic factor expression, β 2 ‐microglobulin expression was used as a control for RNA integrity. cDNA was amplified for the different angiogenic factors with a cycle number known to be located in the linear part of the amplification cycle versus signal intensity curves (De Lange et al , 1997). The signals for the different angiogenic factors were normalized against β 2 ‐microglobulin.…”

mentioning

confidence: 99%

Increased bone marrow vascularization in patients with acute myeloid leukaemia: a possible role for vascular endothelial growth factor

Bont

Rosati²,

Jacobs

et al. 2001

Br J Haematol

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The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0·019) and was restored to normal levels after achieving complete remission (P = 0·03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively. The degree of in vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic factors produced by AML cells. The expression of different angiogenic factors was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial growth factor (VEGF) expression, 4/19 expressed varied spontaneous blastic fibroblast growth factor mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)‐2 and/or MMP‐9. VEGF mRNA expression correlated well with protein level (P = 0·006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0·448, P = 0·05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the growth advantage of the malignant counterpart as a result of the paracrine production of growth factors produced by the surrounding endothelial cells.

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“…On the basis of the extensive genetic studies of AIP, genomic analyses have also shown that PBGD is a pseudogenefree, single-copy gene, thus making it a useful control for RNA analyses. [9][10][11][12][13][14] We sought to use PBGD expression as both an erythroid-specific and housekeeping control for our studies of erythroid-cell gene expression patterns. Unexpectedly, our efforts revealed the existence of 2 distinct erythroid-specific PBGD messenger RNA (mRNA) transcripts.…”

mentioning

confidence: 99%

Human erythroid porphobilinogen deaminase exists in 2 splice variants

Gubin

Miller

2001

Blood

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IntroductionThe third enzyme of the heme synthesis pathway, porphobilinogen deaminase (PBGD), is expressed as 2 isoforms: the so-called "housekeeping" form present in all cell types, and a second isoform expressed only in erythroid cells. [1][2][3][4] The autosomal dominant disorder acute intermittent porphyria (AIP) is caused by mutations in the PBGD gene. 5 Clinically, AIP gene variants may or may not affect enzymatic levels within erythroid cells. 3,6,7 One study reported low erythrocyte PBGD activity in blood donors, whereas lymphocyte PBGD activity was within the normal range. 8 A possible mutation within the erythroid-specific part of the PBGD gene was proposed to explain this finding, but was not further explored. On the basis of the extensive genetic studies of AIP, genomic analyses have also shown that PBGD is a pseudogenefree, single-copy gene, thus making it a useful control for RNA analyses. [9][10][11][12][13][14] We sought to use PBGD expression as both an erythroid-specific and housekeeping control for our studies of erythroid-cell gene expression patterns. Unexpectedly, our efforts revealed the existence of 2 distinct erythroid-specific PBGD messenger RNA (mRNA) transcripts. Study designReverse transcriptase-polymerase chain reaction Total RNA was purified from CD34 ϩ cells collected on specified days after growing in erythropoietin-supplemented media using TRIzol reagent (Life Technologies, Rockville, MD) according to the manufacturer's directions. Reverse transcription (RT) was performed using oligo-(dT) primer and Superscript Reverse Transcriptase (Life Technologies) as suggested by the manufacturer at 42°C for 50 minutes. One microliter of RT reaction was used in 50 L polymerase-chain reaction (PCR) amplification with appropriate primers as follows: initial denaturation at 95°C for 1 minute, denaturing at 95°C for 15 seconds, annealing at 68°C for 15 seconds, and extension at 72°C for 20 seconds for 28 cycles. PCR products were separated in 1.2% agarose gel and stained with ethidium bromide. For PBGD amplification, the following primers were used: 5Ј-ACACAGC-CTACTTTCCAAGCGGAGCCAT-3Ј (primer 1), 5Ј-TGGATCCCTGAG-GAGGGCAGAAG-3Ј (primer 2), 5Ј-TCTTGTCCCCTGTGGTGGACAT-AGCAAT-3Ј (primer 3). Primers 1 and 3 specifically amplify the housekeeping PBGD. Primers 2 and 3 specifically amplify an erythroid PBGD. The PCR products were completely sequenced using a dRhodamine DNA sequencing kit (PE Biosystems, Foster City, CA). Northern blottingHuman Immune System Multiple Tissue Northern (MTN) Blot II was processed as recommended by the manufacturer (Clontech, Palo Alto, CA). Briefly, the membrane was prehybridized in ULTRAhyb hybridization buffer (Ambion, Austin, TX) at 42°C for 30 minutes. The buffer was replaced with the fresh ULTRAhyb containing 32 P-labeled probe and hybridized at 42°C for 16 hours. The membrane was washed in low-and high-stringency buffers (Ambion) according to the manufacturer's protocol and subjected to autoradiography. Probes specific for the housekeeping (probe H) and an erythroid a...

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A Method to Monitor mRNA Levels in Human Breast Tumor Cells Obtained by Fine-needle Aspiration

Cited by 7 publications

References 14 publications

Quantification of telomerase activity, porphobilinogen deaminase and human telomerase reverse transcriptase mRNA in testicular tissue – new parameters for a molecular diagnostic classification of spermatogenesis disorders

Quantification of telomerase activity, porphobilinogen deaminase and human telomerase reverse transcriptase mRNA in testicular tissue – new parameters for a molecular diagnostic classification of spermatogenesis disorders

Increased bone marrow vascularization in patients with acute myeloid leukaemia: a possible role for vascular endothelial growth factor

Human erythroid porphobilinogen deaminase exists in 2 splice variants

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