An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR.

McCulloch, Ross K.; Choong, Catherine S.; Hurley, David M.

doi:10.1101/gr.4.4.219

Cited by 113 publications

(66 citation statements)

References 9 publications

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“…Similar results, showing that shorter fragments amplify more efficiently in quantitative PCR, have been reported earlier. 27 The analysis of amplification efficiencies prevented the underestimation of LDLR mRNA levels in this study: a denaturation temperature of 94°C was initially used for LDLR PCRs, but after poor amplification efficiency of the target became evident, denaturation was performed at 96°C and amplification became efficient. The inefficient amplification of the LDLR target under the initial conditions was probably due to a high GC content of the LDLR gene (69% for the amplified fragment), which makes denaturation of the doublestranded DNA at 94°C incomplete.…”

Section: Competitive Pcrmentioning

confidence: 99%

Expression of LDL Receptor, VLDL Receptor, LDL Receptor–Related Protein, and Scavenger Receptor in Rabbit Atherosclerotic Lesions

et al. 1998

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Background-Atherosclerotic lesions contain foam cells that arise from monocyte-macrophages and smooth muscle cells (SMCs) by excessive uptake of lipoproteins. There are many candidate receptors for the lipid accumulation, such as LDL receptor (LDLR), VLDL receptor (VLDLR), LDL receptor-related protein (LRP), and scavenger receptors (SRs). However, little quantitative information exists on the expression of these receptors in normal and atherosclerotic arteries. Methods and Results-Competitive reverse transcription-polymerase chain reaction and in situ hybridization were used for the studies in New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbit aortic intima-medias. NZW rabbits were fed a 1% cholesterol diet for 0 (control group), 3, 6, or 14 weeks. LDLR mRNA expression was low in aortic intima-medias of all groups. Of the analyzed receptors, LRP had the highest expression in the control group, and its mRNA was induced threefold in the 14-week group, the aortas of which had extensive lesions. SR expression was low and VLDLR expression moderate in the control group. Both receptors were highly induced during cholesterol feeding (SRs, 3-fold and 270-fold induction; VLDLR, 15-fold and 100-fold induction in the 3-week and 14-week groups, respectively). Comparable results were obtained from WHHL rabbits: high basal LRP mRNA in normal intima-medias; moderate induction of LRP and marked induction of SRs and VLDLR in fatty streaks and fatty plaques. In situ hybridization indicated that LRP and VLDLR were expressed in SMCs and macrophages. VLDLR expression was also observed in endothelial cells. SR expression was detected only in macrophages. Conclusions-SR and VLDLR mRNAs were highly induced in atherosclerotic lesions. VLDLR and LRP may be involved in the formation of both SMC-and macrophage-derived foam cells, whereas SRs play an important role in lipid uptake in macrophages.

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Section: Competitive Pcrmentioning

confidence: 99%

Expression of LDL Receptor, VLDL Receptor, LDL Receptor–Related Protein, and Scavenger Receptor in Rabbit Atherosclerotic Lesions

et al. 1998

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“…However, it is generally agreed that the most important factor determining overall amplification is related to the specific primers used. 5,14 Considering these factors, the IBD IC was designed to have priming sites identical to, and be larger in size than, the IBDV target template. Because the sequence of the control was required to be free of the restriction sites used in our RFLP analysis, the use of a modified (deletion/insertion) IBD genome segment was not practical.…”

Section: Discussionmentioning

confidence: 99%

Development of a ssRNA Internal Control Reagent for an Infectious Bursal Disease Virus Reverse Transcription/Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Diagnostic Assay

1999

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Abstract. Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens. A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay. An 841-bp bacteriophagelambda DNA fragment was directionally ligated to 3Ј and 5Ј oligonucleotide linkers containing the IBDV RT/ PCR target primer sequences. A pGEM-3Zf (ϩ) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA. After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product. The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms. With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay. By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results.Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, 3 is the causative agent of an economically important immunosuppressive disease of young chickens. The use of the reverse transcription/ polymerase chain reaction (RT/PCR) to detect IBDV in field and laboratory samples has been reported previously. 7,11,12,17,18,21 A RT/PCR diagnostic assay for IBDV was developed in this laboratory. 8 In the assay, 743-bp RT/PCR products containing the coding sequence for a portion of the IBDV structural protein VP2 are digested with the restriction enzymes (REs) BstNI and MboI to yield a variety of restriction fragment length polymorphism (RFLP) patterns. These patterns are used to place field, vaccine, and previously characterized laboratory strains of the virus into different molecular groups. 7,9 The application of PCR and RT/PCR methodologies in research and diagnostic testing has become commonplace since the first description of the PCR by Saiki. 16 Although these methods have led to the development of powerful diagnostic assays with respect to observed and theoretical sensitivity and specificity, they are also quite complex. A number of factors can influence the success or failure of an indi- However, the performance of individual PCR and RT/ PCR tests can vary from 1 sample to another because of the presence of inhibitors. 4,15,19 The tissue type and the quality of extracted RNA have been noted to affect test performance. As an aide in monitoring reaction performance, and to identify the occurrence of false-negative tests due to reaction inhibition or technical errors in test preparation, a RE assay-compatible IC reagent was developed. The production and use of this IC is described.

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“…Care should be taken when manipulating the size of the competitors because it could greatly influence the amplification efficiency. 56,57 Multispecific competitors, containing targets for several pairs of primers, corresponding to several GMOs, have also been designed, 58,59 but this technique should face the problem of the different amplification efficiencies of the target sequences with that of the common competitor.…”

Section: Qc-pcrmentioning

confidence: 99%

Detection of Genetically Modified Organisms in Foods by DNA Amplification Techniques

García‐Cañas

Cifuentes

González

2004

Critical Reviews in Food Science and Nutrition

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An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR.

Cited by 113 publications

References 9 publications

Expression of LDL Receptor, VLDL Receptor, LDL Receptor–Related Protein, and Scavenger Receptor in Rabbit Atherosclerotic Lesions

Expression of LDL Receptor, VLDL Receptor, LDL Receptor–Related Protein, and Scavenger Receptor in Rabbit Atherosclerotic Lesions

Development of a ssRNA Internal Control Reagent for an Infectious Bursal Disease Virus Reverse Transcription/Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Diagnostic Assay

Detection of Genetically Modified Organisms in Foods by DNA Amplification Techniques

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