scholarly journals Expression of the Multidrug Resistance-Associated Protein Gene in Refractory Lymphoma: Quantitation by a Validated Polymerase Chain Reaction Assay

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3795-3800 ◽  
Author(s):  
Zhirong Zhan ◽  
Victor A. Sandor ◽  
Erick Gamelin ◽  
Joanna Regis ◽  
Bruce Dickstein ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multidrug Resistance ◽  
Mrna Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Newly Diagnosed ◽  
Chain Reaction ◽  
Significant Difference ◽  
Polymerase Chain ◽  
Refractory Lymphoma ◽  
Mdr 1

Abstract Previous work investigating the role of MDR-1 overexpression in relapsed and refractory lymphoma led us to investigate a possible role for multidrug resistance-associated protein (MRP) as a cause of resistance in patients who did not overexpress MDR-1. A quantitative polymerase chain reaction (PCR) method for measuring MRP expression was validated. Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein levels. MRP levels were found to be independent of sample tumor content by immunophenotyping, suggesting that the presence of normal cells had no significant impact on measurements of MRP expression. We evaluated MRP in 55 biopsy samples from 40 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH). Pre- and post-EPOCH samples were available from 15 patients. MRP levels were also evaluated in 16 newly diagnosed, untreated lymphoma patient samples. No significant difference in MRP mRNA expression was noted between pre- and post-EPOCH groups. Also, MRP levels in the newly diagnosed patient samples were not significantly different from either pre- or post-EPOCH groups. Two of 15 paired pre- and post-EPOCH patient samples exhibited overexpression of MRP after EPOCH chemotherapy, with measured increases of 10-fold and 18-fold. We conclude that MRP overexpression is not responsible for non–P-glycoprotein (Pgp)–mediated drug resistance in the majority of these patients, although it may be important in a subset of patients. Defining this subset prospectively could aid in the development of clinical trials of MRP modulation in drug-resistant lymphoma.

Expression of the Multidrug Resistance-Associated Protein Gene in Refractory Lymphoma: Quantitation by a Validated Polymerase Chain Reaction Assay

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3795-3800 ◽  
Author(s):  
Zhirong Zhan ◽  
Victor A. Sandor ◽  
Erick Gamelin ◽  
Joanna Regis ◽  
Bruce Dickstein ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multidrug Resistance ◽  
Mrna Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Newly Diagnosed ◽  
Chain Reaction ◽  
Significant Difference ◽  
Polymerase Chain ◽  
Refractory Lymphoma ◽  
Mdr 1

Previous work investigating the role of MDR-1 overexpression in relapsed and refractory lymphoma led us to investigate a possible role for multidrug resistance-associated protein (MRP) as a cause of resistance in patients who did not overexpress MDR-1. A quantitative polymerase chain reaction (PCR) method for measuring MRP expression was validated. Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein levels. MRP levels were found to be independent of sample tumor content by immunophenotyping, suggesting that the presence of normal cells had no significant impact on measurements of MRP expression. We evaluated MRP in 55 biopsy samples from 40 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH). Pre- and post-EPOCH samples were available from 15 patients. MRP levels were also evaluated in 16 newly diagnosed, untreated lymphoma patient samples. No significant difference in MRP mRNA expression was noted between pre- and post-EPOCH groups. Also, MRP levels in the newly diagnosed patient samples were not significantly different from either pre- or post-EPOCH groups. Two of 15 paired pre- and post-EPOCH patient samples exhibited overexpression of MRP after EPOCH chemotherapy, with measured increases of 10-fold and 18-fold. We conclude that MRP overexpression is not responsible for non–P-glycoprotein (Pgp)–mediated drug resistance in the majority of these patients, although it may be important in a subset of patients. Defining this subset prospectively could aid in the development of clinical trials of MRP modulation in drug-resistant lymphoma.

scholarly journals Expression of mdr-1 in refractory lymphoma: quantitation by polymerase chain reaction and validation of the assay [published erratum appears in Blood 1995 Dec 15;86(12):4710]

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1515-1524 ◽  
Author(s):  
YK Kang ◽  
Z Zhan ◽  
J Regis ◽  
M Alvarez ◽  
R Robey ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
T Cells ◽  
Chain Reaction ◽  
P Glycoprotein ◽  
Polymerase Chain ◽  
Beta 2 ◽  
Refractory Lymphoma ◽  
Beta 2 Microglobulin ◽  
The Impact ◽  
Mdr 1

Measurement of P-glycoprotein and the gene that encodes it, mdr-1, is an important tool for assessing the impact of multidrug resistance in clinical cancer. We evaluated mdr-1 expression by a quantitative polymerase chain reaction (PCR) assay in 78 biopsy samples from 48 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH) in which R-verapamil was added as an antagonist of P-glycoprotein in a subset of patients whose tumors were unresponsive to treatment. Expression of mdr-1 was detectable in all biopsies at the time of enrollment on study, and a fourfold or greater increase in mdr- 1 expression was noted in 42% of patients at the time of treatment failure. Expression of mdr-1 was also detectable in biopsies from patients at the time of diagnosis of lymphoma. An endogenous control gene, beta 2-microglobulin, was quantitated for normalization of the mdr-1 values. The use of beta 2-microglobulin expression for normalization was validated in a subset of samples by comparing Northern blots detecting beta 2-microglobulin, beta actin, and GAPDH gene expression. Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein level. Immunophenotyping of lymphomatous lymph nodes showed that infiltration of tumor cells ranged from 8% to 95% and of normal T cells from 1% to 83%. Expression of mdr-1 in normal T cells and monocytes was also shown to be low. The mdr-1 levels in patient samples were independent of T- cell contamination, suggesting that the presence of normal cells has at best a small impact on mdr-1 measurements. Expression of mdr-1 in lymphoma can be quantitated by PCR, and wide variations in expression can be observed. Increased expression in patients with refractory disease supports an important role for Pgp in drug resistance in lymphoma. These studies will aid in the design and interpretation of clinical trials in lymphoma.

Evaluation of quantitative polymerase chain reaction for the detection of Toxoplasma gondii oocysts shed by cats

2021 ◽  
Vol 30 (4) ◽  
Author(s):  
Ana Carolina Miura ◽  
Luiz Daniel de Barros ◽  
Ana Flávia Minutti ◽  
Thais Agostinho Martins ◽  
João Pedro Sasse ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Toxoplasma Gondii ◽  
Microscopic Examination ◽  
Statistical Difference ◽  
Quantitative Polymerase Chain Reaction ◽  
Post Inoculation ◽  
Chain Reaction ◽  
Significant Difference ◽  
Flotation Technique ◽  
Polymerase Chain

Abstract Felines are definitive hosts of Toxoplasma gondii and can shed oocysts in their feces, contaminating the environment. Sporulated oocysts are highly resistant to the environment and have higher infectivity, which are attributed to many toxoplasmosis outbreaks. The aim of the present study was to evaluate a quantitative polymerase chain reaction (qPCR) technique for the detection of T. gondii oocysts shed by cats. Twelve cats from a previous vaccine experiment were challenged orally with 600 cysts of the TgDoveBr8 strain on day 72. Fecal samples were collected daily using the centrifugal flotation technique, with microscopic examination (Sheather technique) and qPCR for 20 days after the challenge. Cats from all groups shed oocysts in their feces. Five negative cats in the Sheather were positive according to qPCR on the 3rd day post-inoculation (dpi). Oocysts were detected on the 4th dpi using the Sheather; however, there was no statistical difference between the two methods (p=0.1116). In addition, there was no statistically significant difference in oocyst shedding between the groups according to the Sheather technique (p=0.6534) and qPCR (p=0.9670). In conclusion, these results demonstrate that qPCR can be used as an alternative to the Sheather to detect and quantify T. gondii oocysts.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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