Quantification of G protein mRNA using reverse transcription and competitive PCR with a colorimetric microplate assay

1998 ◽  
Vol 12 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Rupa Mokkapatti ◽  
Richard A Conn ◽  
Joseph A Carcillo ◽  
Marjorie Romkes ◽  
Edwin K Jackson
Keyword(s):  
G Protein ◽  
Reverse Transcription ◽  
Microplate Assay ◽  
Competitive Pcr

scholarly journals IL-1α, IL-1β, IL-6, and TNF-α Steady-State mRNA Levels Analyzed by Reverse Transcription-Competitive PCR in Bone Marrow of Gonadectomized Mice

1998 ◽  
Vol 13 (2) ◽  
pp. 185-194 ◽  
Author(s):  
Rutger L. Van Bezooijen ◽  
Hetty C. M. Farih-Sips ◽  
Socrates E. Papapoulos ◽  
Clemens W. G. M. Löwik
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Reverse Transcription ◽  
Mrna Levels ◽  
Competitive Pcr ◽  
Tnf Α

Quantitative Analysis of Constitutive and Inducible CYPs mRNA Expression in the HepG2 Cell Line Using Reverse Transcription-Competitive PCR

2000 ◽  
Vol 267 (3) ◽  
pp. 756-760 ◽  
Author(s):  
Akihiko Sumida ◽  
Shuichi Fukuen ◽  
Isamu Yamamoto ◽  
Hideyasu Matsuda ◽  
Masakazu Naohara ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Mrna Expression ◽  
Cell Line ◽  
Hepg2 Cell ◽  
Reverse Transcription ◽  
Hepg2 Cell Line ◽  
Competitive Pcr

scholarly journals Clinical Evaluation of an In-House Reverse Transcription-Competitive PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

1999 ◽  
Vol 37 (2) ◽  
pp. 333-338 ◽  
Author(s):  
Maurizio Zazzi ◽  
Laura Romano ◽  
Marinunzia Catucci ◽  
Giulietta Venturi ◽  
Angelo De Milito ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Clinical Samples ◽  
Reaction Products ◽  
Competitive Pcr ◽  
Immunodeficiency Virus ◽  
Hiv 1

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma samples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitive PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reaction products, and (v) densitometric analysis of the digitized image of the gel. Quadruplicate tests and dilution studies showed that the sensitivity and intertest coefficient of variability of the RT-cPCR are comparable to those of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mean difference, 0.36 ± 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA levels with the expected inverse correlation between CD4 counts and HIV-1 RNA titers. In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia. The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (P = 0.0210), 1.584 log units for those receiving indinavir (P = 0.0047), and 1.904 log units for those receiving ritonavir (P < 0.0001). The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 to 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.

Simple, sensitive and accurate method for the quantification of prothrombin mRNA by using competitive PCR

2001 ◽  
Vol 356 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Phulwinder K. GROVER ◽  
Alan M. F. STAPLETON ◽  
Katsuhito MIYAZAWA ◽  
Rosemary Lyons RYALL
Keyword(s):  
Reverse Transcription ◽  
Accurate Method ◽  
Competitive Pcr ◽  
Tissue Samples ◽  
Total Rna ◽  
Standard Curve ◽  
Minimum Number ◽  
Identical Nucleotide ◽  
Number Of Cycles ◽  
Made In

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or β-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to β-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean±S.D. of 0.37±0.07 (range 0.27–0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.

Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma

2000 ◽  
Vol 87 (1-2) ◽  
pp. 91-97 ◽  
Author(s):  
Giulietta Venturi ◽  
Rebecca Ferruzzi ◽  
Laura Romano ◽  
Marinunzia Catucci ◽  
Pier E Valensin ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Competitive Pcr ◽  
Hiv 1

scholarly journals Flow Cytometric Quantification of Competitive Reverse Transcription-PCR Products

2002 ◽  
Vol 48 (9) ◽  
pp. 1398-1405 ◽  
Author(s):  
Niels Wedemeyer ◽  
Thomas Pötter ◽  
Steffi Wetzlich ◽  
Wolfgang Göhde
Keyword(s):  
Internal Control ◽  
Reverse Transcription ◽  
Specific Binding ◽  
Human Lymphocytes ◽  
Fluorescence Labeling ◽  
Target Sequence ◽  
Competitive Pcr ◽  
Rt Pcr ◽  
Flow Cytometric ◽  
Pcr Products

Abstract Background: Competitive PCR of reverse transcribed mRNA sequences is used to quantify transcripts, but the usual approaches are labor-intensive and time-consuming. We describe the non-gel-based quantification of competitive reverse transcription (RT)-PCR products with use of microparticles and flow cytometry. Methods: PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively. Results: As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half. Conclusions: The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method.

Relationship between mRNA Levels Quantified by Reverse Transcription-Competitive PCR and Metabolic Activity of CYP3A4 and CYP2E1 in Human Liver

1999 ◽  
Vol 262 (2) ◽  
pp. 499-503 ◽  
Author(s):  
Akihiko Sumida ◽  
Kayoko Kinoshita ◽  
Tsuyoshi Fukuda ◽  
Hideyasu Matsuda ◽  
Isamu Yamamoto ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Reverse Transcription ◽  
Human Liver ◽  
Mrna Levels ◽  
Competitive Pcr

scholarly journals A Simple Quantitative Measurement of mRNA of Human .BETA.-actin by Reverse Transcription Competitive PCR with a Compact Digital Camera.

1997 ◽  
Vol 20 (9) ◽  
pp. 1013-1016 ◽  
Author(s):  
Yoshihiro OKAMOTO ◽  
Yusuke HARA ◽  
Yayoi KATOH ◽  
Hiroshi NAGAI ◽  
Mikio NISHIDA
Keyword(s):  
Reverse Transcription ◽  
Quantitative Measurement ◽  
Digital Camera ◽  
Competitive Pcr ◽  
Beta Actin
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