Rapid Development of a Quantitative-Competitive (qc) RT-PCR Assay Using a Composite Primer Approach

O’Connell, Joe; Houston, Aileen; Kelly, Raymond; O'Brien, D. P.; Ryan, Aideen; Bennett, Michael; Nally, Kenneth

doi:10.1385/1-59259-283-x:093

Cited by 2 publications

(5 citation statements)

References 2 publications

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“…On one hand the competitor product has to be distinguishable from the native template but at the same time too much of difference in their size may cause differences in amplification efficiency. As a solution to handle with this problem it is generally recommended that the size difference between the target and competitor product should not be more than 30% (O'Connell et al. 2002).…”

Section: Discussionmentioning

confidence: 99%

“…2000). However, in competitive PCR because two templates (target and competitor) compete for PCR amplification it may be necessary to perform PCR for more number of cycles to achieve good amplification of both templates specially when the target copy number is very low and at the lower limit of detection (O'Connell et al. 2002).…”

Section: Discussionmentioning

confidence: 99%

“…2002). For carrying out the competitive quantitative PCR, a composite forward primer (henceforth called 18s CF primer) was designed as previously reported by O'Connell et al. (2002).…”

Section: Methodsmentioning

confidence: 99%

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Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

Aswal

Datta

Raghav

et al. 2007

Reprod Domestic Animals

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The present work describes the development of a quantitative competitive PCR strategy for quantifying the relative abundance of 18s rRNA transcripts in buffalo oocytes during in vitro maturation (IVM). As a method, the competitive PCR overcomes some of the shortcomings of conventional reverse transcriptase polymerase chain reaction (RT-PCR) procedure making it a more authentic quantitative method. A composite primer based approach was used to generate the competitor cDNA to be used as external control. Validity of the method for its efficiency was demonstrated by quantitative analysis of the competition parameters. Using this method the relative abundance of buffalo oocyte 18s rRNA transcript over the period of IVM was found to vary within a narrow range of 0.93-1.06 folds which establishes the accuracy of the method and reflects the stability of its expression during IVM. This qualifies the use of this house keeping gene as a valid internal control in studies investigating the gene expression pattern in buffalo oocytes. The competitive PCR approach described in this study could be used for quantification of other transcripts from a limited number of oocytes where a conventional RT-PCR method is either difficult to use or multiplexing it with highly abundant house keeping genes is apparently problematic.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

Aswal

Datta

Raghav

et al. 2007

Reprod Domestic Animals

View full text Add to dashboard Cite

show abstract

“…The plasmid thus obtained (pBS/competitor A + B) contained competitors for AML1a and AML1b in back-to-back orientation, with 1,250 bp of intervening spacer. This pBS/competitor A + B was used for the competitive PCR for both AML1a and AML1b/AML1 c to quantitate AML1 isoforms in a given cell population [34]. Total RNA was isolated from the fractionated cord blood cells using Trizol reagent (Invitrogen, http://www.invitrogen.com) and then reverse-transcribed using random primers and Superscript II enzyme according to the manufacturers' instructions.…”

Section: Methodsmentioning

confidence: 99%

“…PCR products were then fractionated on a 2.5% agarose gel electrophoresis, and the PCR products from endogenous AML1 and from the competitor plasmid were quantitated densitometrically. The amounts of the endogenous AML1 isoforms were then estimated as described [34] and normalized on the basis of G6PD expression estimated by a quantitative real-time PCR [35]. …”

Section: Methodsmentioning

confidence: 99%

Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1

et al. 2007

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Background AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.Methods and FindingsThe human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.ConclusionsThese data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.

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Rapid Development of a Quantitative-Competitive (qc) RT-PCR Assay Using a Composite Primer Approach

Cited by 2 publications

References 2 publications

Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1

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