Real-Time Homogeneous Assay of Rapid Cycle Polymerase Chain Reaction Product for Identification ofLeptonema illini

Woo, Tony H.S.; Patel, Bharat K. C.; Cinco, Marina; Smythe, Lee D.; Symonds, Meegan L.; Norris, Michelle A.; Dohnt, Michael F.

doi:10.1006/abio.1997.2532

Cited by 35 publications

(21 citation statements)

References 15 publications

(9 reference statements)

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“…43 No specific probes were used in our study, and SYBR Green I dye that bound to double-stranded DNA and provided the fluorescent signal was used. 44,45 This approach is simpler, cheaper and probably more sensitive, since many fluorescent labels, instead of just 1 molecule, incorporated into amplified fragment. The disadvantage of fluorescence dye is that specific and non-specific products generate signal.…”

Section: Discussionmentioning

confidence: 99%

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Tanaka

Kobayashi

Utsuki

et al. 2002

Intl Journal of Cancer

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Key words: MGMT; real-time RT-PCR; ACNU; gliomaNitrosoureas are alkylating agents that cause cell death by binding to DNA. Among nitrosoureas, 1-(4-amino-2-methyl-5-pyrimidynyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) is widely used in Japan to treat gliomas because of its ability to permeate the blood-brain-barrier and excellent clinical effects in combination with radiation and interferon (IFN)-␤. [1][2][3] Drug-resistance genes are some of the most important elements of tumors themselves in determining drug-resistance, 4 and O 6 -methylguanine-DNA methyltransferase (MGMT) is a drug-resistance gene for nitrosoureas. 5,6 The cross-linking of doublestranded DNA by alkylating agents is inhibited by the cellular DNA-repair protein MGMT. MGMT rapidly reverses alkylation at the O 6 position of guanine, thereby averting lethal cross-linking. 7 This is the mechanism by which MGMT induces resistance to alkylating agents such as ACNU.Protocol studies for malignant gliomas have not provided encouraging therapeutic results because of the heterogeneity and various drug-sensitivities of the tumors. 8,9 Individualization of glioma therapy is recommended. We carried out a new adjuvant therapy that was individualized based on the results of the reverse transcription-polymerase chain reaction (RT-PCR) for MGMT to treat malignant gliomas. 2 Our preliminary individual adjuvant therapies (IAT) based on RT-PCR results regarding MGMT expression seemed to be more effective than conventional therapies for malignant gliomas.The level of MGMT varies widely according to the type of tumor, and even varies among tumors of the same histological classification. 5,10 Approximately 30 -50% of gliomas lack MGMT. 11 The fact that more than 50% of gliomas express MGMT limits the indications for ACNU. In our preliminary IAT, platinum (Pt)-compounds were used instead of ACNU even though Ptcompounds had not been accepted as being effective in gliomas. 12,13 To ensure IAT for gliomas, quantitative evaluation is needed for a higher initial response and prolongation of survival.We carried out the present study to determine more appropriate indications for the use of ACNU in gliomas. Real-time quantitative RT-PCR was used to re-evaluate the treated gliomas. MATERIAL AND METHODS Real-time quantitative RT-PCRIn 100 frozen sample of neuroepithelial tumors (19 low-grade neuroepithelial tumors, 28 Grade III gliomas, 41 glioblastomas, and 12 medulloblastomas), MGMT was quantitated by real-time quantitative RT-PCR using SYBR Green dye. RT-PCR was basically carried out as described previously. 14 -16 Briefly, total RNA was extracted from frozen tissue specimens weighing about 1 g, which had been homogenized using a glass Teflon homogenizer, via the guanidinium thiocyanate-phenol-chloroform single-step extraction method with Isogen (Nippon Gene, Toyama, Japan). 17 After ethanol precipitation, about 100 g of total RNA was extracted. Forty microliters of complementary deoxyribonucleic acid (cDNA) solution was synthesized from 2 g of total RNA wi...

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Section: Discussionmentioning

confidence: 99%

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Tanaka

Kobayashi

Utsuki

et al. 2002

Intl Journal of Cancer

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“…A standard curve for PCR test was constructed using serial 1:5 dilutions of normal murine testis cDNA. The concentrations of samples, analyzed in triplicate, were determined with the help of standard curve using the fit-points method in data-analysis software (Wittwer et al 1997, Woo et al 1998. The mean of two repeated PCR values was used in the statistical analyses.…”

Section: Rt (Reverse Transcriptase)-pcrmentioning

confidence: 99%

“…Results were normalized to porphobilinogen deaminase (PBGD) by dividing the individual RT-PCR values by the mean of three repeated PBGD test values of the respective sample to reduce variability between RNA amounts introduced into the RT-PCR reactions (Schrader et al 2002). To distinguish the specific PCR products from non-specific products and primer dimers, a melting-curve analysis was performed as described earlier (Woo et al 1998). Additionally, samples were analyzed by agarose gel electrophoresis to verify that the amplified products exhibited correct sizes.…”

Section: Rt (Reverse Transcriptase)-pcrmentioning

confidence: 99%

Disruption of the murine PIASx gene results in reduced testis weight

Santti

Mikkonen²,

Anand³

et al. 2005

Journal of Molecular Endocrinology

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PIASx belongs to the PIAS protein family, the members of which modulate activities of several transcription factors and act as E3 ligases in the sumoylation pathway. The PIASx gene is highly expressed in testis, suggesting a role in spermatogenesis. To investigate the function of PIASx in vivo, we have disrupted the PIASx gene in mice. Interestingly, the knockout mice were viable and fertile. Despite the normal fertility, the testis weight of the mutant animals was reduced and their number of apoptotic testicular cells was increased. Also, the sperm count of mutant mice tended to be reduced, but the quality of their sperm cells was normal. No significant changes were observed in the serum levels of LH and FSH or in the intratesticular testosterone concentration between the knockout animals and their wild-type littermates. Compensatory increases in other PIAS protein mRNAs were not observed in the knockout mice. These results imply that PIASx is required quantitatively rather than qualitatively for normal spermatogenesis.

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“…For distinguishing specific from non-specific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 708C for 30 s followed by a gradual increase of temperature to 988C at a rate of 0.28C/s, with the signal acquisition mode set at step, as described before (Woo et al, 1998). To verify the melting curve results, representative samples of the PCR products were run on 1.5% agarose gels, purified, and cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.…”

Section: Melting Curvementioning

confidence: 99%

Human kallikrein gene 13 (KLK13) expression by quantitative RT–PCR: an independent indicator of favourable prognosis in breast cancer

et al. 2002

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Kallikreins are a group of serine proteases with diverse physiological functions. KLK13 (previously known as KLK-L4) is a novel kallikrein gene located on chromosome 19q13.4 and shares a high degree of homology with other kallikrein family members. Many kallikrein genes were found to be differentially expressed in various malignancies, and their regulation is controlled by steroid hormones in prostate and breast cancer cell lines. We studied the expression of KLK13 by quantitative reverse transcriptase -polymerase chain reaction in 173 patients with epithelial breast carcinoma. An optimal cutoff point equal to the 40th percentile was defined, based on the ability of KLK13 to predict disease-free survival. KLK13 values were then associated with other established prognostic factors and with disease-free survival and overall survival. Higher positivity for KLK13 expression was found in older, oestrogen receptor positive patients. In univariate analysis, KLK13 expression is a significant predictor of improved disease-free survival and overall survival (P50.001 and P=0.009, respectively). Cox multivariate analysis indicated that KLK13 was an independent prognostic variable in the subgroups of patients with Grade I -II tumours and in patients who were oestrogen receptor and progesterone receptor positive, and node positive. Hazard ratios derived from Cox analysis, related to disease-free survival and overall survival were 0.22 (P=0.001) and 0.24 (P=0.008), respectively, for the Grade I -II group; 0.36 (P=0.008) and 0.44 (P=0.038), respectively, for the node positive group and 0.36 (P=0.008) and 0.18 (P=0.008), respectively, for the oestrogen receptor positive group. The adjusted hazard ratio for progesterone receptor positive patients for disease-free survival was 0.25 (P=0.012). For patients in the node positive and oestrogen receptor positive subgroup (n=51) the adjusted hazard ratio was 0.25 (P=0.006) and for the node positive and progesterone receptor positive subgroup (n=46) the hazard ratio was 0.24 (P=0.008). Taken together, these data suggest that higher KLK13 expression in these subgroups of breast cancer patients is associated with an approximately 55 to 80% reduction in the risk of relapse or death. We conclude that KLK13 expression, as assessed by quantitative reverse transcriptase -polymerase chain reaction, is an independent favourable prognostic marker for breast carcinoma.

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Real-Time Homogeneous Assay of Rapid Cycle Polymerase Chain Reaction Product for Identification ofLeptonema illini

Cited by 35 publications

References 15 publications

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Disruption of the murine PIASx gene results in reduced testis weight

Human kallikrein gene 13 (KLK13) expression by quantitative RT–PCR: an independent indicator of favourable prognosis in breast cancer

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Real-Time Homogeneous Assay of Rapid Cycle Polymerase Chain Reaction Product for Identification ofLeptonema illini

Cited by 35 publications

References 15 publications

O6‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

O6‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Disruption of the murine PIASx gene results in reduced testis weight

Human kallikrein gene 13 (KLK13) expression by quantitative RT–PCR: an independent indicator of favourable prognosis in breast cancer

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Product

Resources

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O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas