Technical Note
Multiplex preamplification of specific cDNA targets prior to gene expression analysis by TaqMan Arrays
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* Corresponding author: Lourdes Mengual lmengual@ub.edu
1 Laboratory and Department of Urology. Institut Clínic de Nefrologia i Urologia (ICNU), Hospital Clínic de Barcelona. Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Villarroel, 170, 08036 Barcelona, Spain
2 Molecular Biology Laboratory. Fundació Puigvert. Universitat Autònoma de Barcelona, Cartagena 340-350, 08025 Barcelona, Spain
BMC Research Notes 2008, 1:21 doi:10.1186/1756-0500-1-21
Published: 5 June 2008Abstract
Background
An accurate gene expression quantification using TaqMan Arrays (TA) could be limited by the low RNA quantity obtained from some clinical samples. The novel cDNA preamplification system, the TaqMan PreAmp Master Mix kit (TPAMMK), enables a multiplex preamplification of cDNA targets and therefore, could provide a sufficient amount of specific amplicons for their posterior analysis on TA.
Findings
A multiplex preamplification of 47 genes was performed in 22 samples prior to their analysis by TA, and relative gene expression levels of non-preamplified (NPA) and preamplified (PA) samples were compared. Overall, the mean cycle threshold (CT) decrement in the PA genes was 3.85 (ranging from 2.07 to 5.01). A high correlation (r) between the gene expression measurements of NPA and PA samples was found (mean r = 0.970, ranging from 0.937 to 0.994; p < 0.001 in all selected cases). High correlation coefficients between NPA and PA samples were also obtained in the analysis of genes from degraded RNA samples and/or low abundance expressed genes.
Conclusion
We demonstrate that cDNA preamplification using the TPAMMK before TA analysis is a reliable approach to simultaneously measure gene expression of multiple targets in a single sample. Moreover, this procedure was validated in genes from degraded RNA samples and low abundance expressed genes. This combined methodology could have wide applications in clinical research, where scarce amounts of degraded RNA are usually obtained and several genes need to be quantified in each sample.