Determination of Dihydrofolate Reductase Gene Amplification from Single Cell Colonies by Quantitative Polymerase Chain Reaction

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay.

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Detection of a Single Base Mutation in the Human Dihydrofolate Reductase Gene from a Methotrexate-Resistant Cell Line Using the Polymerase Chain Reaction

Cancer Communications ◽

10.3727/095535489820875453 ◽

1989 ◽

Vol 1 (1) ◽

pp. 7-12 ◽

Cited By ~ 6

Author(s):

Adam P. Dicker ◽

Matthias Volkenandt ◽

Joseph R. Bertino

Keyword(s):

Polymerase Chain Reaction ◽

Cell Line ◽

Dihydrofolate Reductase ◽

Resistant Cell ◽

Resistant Cell Line ◽

Chain Reaction ◽

Single Base ◽

Reductase Gene ◽

Polymerase Chain ◽

Human Dihydrofolate Reductase

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Rapid and accurate determination of the potency of varicella vaccine by quantitative polymerase chain reaction

Vaccine ◽

10.1016/j.vaccine.2011.09.017 ◽

2011 ◽

Vol 29 (47) ◽

pp. 8490-8495 ◽

Cited By ~ 1

Author(s):

Marsha S. Russell ◽

Changgui Li ◽

Louise Larocque ◽

Junzhi Wang ◽

Aaron Farnsworth ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Polymerase Chain Reaction ◽

Accurate Determination ◽

Varicella Vaccine ◽

Chain Reaction ◽

Polymerase Chain

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Determination of carrier status in Duchenne and Becker muscular dystrophies by quantitative polymerase chain reaction and allele-specific oligonucleotides

Clinical Chemistry ◽

10.1093/clinchem/36.12.2113 ◽

1990 ◽

Vol 36 (12) ◽

pp. 2113-2117 ◽

Cited By ~ 17

Author(s):

T W Prior ◽

A C Papp ◽

P J Snyder ◽

W E Highsmith ◽

K J Friedman ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Muscular Dystrophy ◽

Gene Dosage ◽

Quantitative Polymerase Chain Reaction ◽

Becker Muscular Dystrophy ◽

Carrier Status ◽

Chain Reaction ◽

Allele Specific ◽

Polymerase Chain

Abstract Detection of carriers of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), in the deletion cases, involves calculating gene dosage from Southern blots. We show that the analysis of dosage can also be made from the polymerase chain reaction (PCR) with use of allele-specific oligonucleotides (ASOs). The deletion-prone exons are amplified, transferred to a membrane, and hybridized with ASOs complementary to the exons; the autoradiographic bands are then quantified with a densitometer. After determining the quantitative conditions of the amplification reaction, we were able to identify deletions in a DMD/BMD carrier female. The determination of carrier status via PCR removes several of the technical limitations of Southern analysis and is also cost- and labor-effective.

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