A Relevant Reference Gene and Normalization for mRNA Real-Time PCR Quantification in Specimens with Distinct Cell Types and Variant Integrity

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Variability of clinical samples with respect to cell types and quality makes it indispensable to normalize mRNA quantification by real-time reverse transcription-polymerase chain reaction (RT-PCR). The objective of the present study was to elucidate the influence of the difference in RNA integrity and the expression status of control genes commonly used. To compare the expression status of GAPDH, β-actin, PBGD and 18S rRNA in different cell samples, real-time RT-PCR by the LightCycler Technology was applied. The relevance of the above-mentioned normalization by control gene was evaluated through the practical measurement of survivin using normal lymphocytes from 19 healthy donors, adult T-cell leukemia (ATL) cells from 30 patients with ATL and 27 cell line cells. The mRNA integrity was found to be tolerable for absolute quantification when the preservation rate of 28S and 18S rRNA within total RNA was at least 30%. The expression status of control genes was prominently variable with the expression difference of 4-fold in β-actin, 20-fold in GAPDH, 30-fold in 18S rRNA and 66-fold in PBGD among the different cell types, normal lymphocytes, chronic and acute ATL cells, ATL cell lines, other hematopoietic cell lines and solid tumor cell lines. The survivin mRNA data normalized by such a control gene were influenced by the expression variability of the control genes. These results indicate that the control genes can be used to correct for sample-to-sample variation within the same cell types, but they are not always relevant for normalization in the distinct cell types.
長崎大学医学部の論文

A Relevant Reference Gene and Normalization for mRNA Real-Time PCR Quantification in Specimens with Distinct Cell Types and Variant Integrity

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長崎大学医学部 | 論文

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