Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.

Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.

Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia.

The p73 protein shares structural and functional similarities with the tumour-suppressor p53, but its role in neoplastic transformation is unknown. Alternative splicing leads to the expression of at least nine p73 C-terminal mRNA splice variants (alpha, beta, gamma, delta, epsilon, zeta, eta, eta1, theta). In this survey, we analyse the expression of p73 by real-time quantitative RT-PCR, its known C-terminal variants with an RT-PCR-Southern technique and by Western blot in samples of 51 patients with B-CLL, normal B lymphocytes from eight individuals, and five haematopoetic cell lines. p73alpha protein expression positively correlated with higher risk B-CLL stages (P = 0.046). Total p73 mRNA expression was higher (P = 0.01) and p73alpha protein more frequently detected (P = 0.008) in B-CLL compared with normal CD19+-B-lymphocytes. p73 C-terminal mRNA variants were expressed both in B-CLL and in normal B-lymphocytes, but their expression was biased since the gamma (P = 0.041), the theta (P < 0.001), and the eta variant (P = 0.033) prevailed in normal B-lymphocytes. In summary, we conclude that the accumulation of p73, the expression pattern of particular p73 variants and its link to progression may play a distinct role in the molecular pathology B-CLL.

Distinct expression patterns of the p53-homologue p73 in malignant and normal hematopoiesis assessed by a novel real-time reverse transcription-polymerase chain reaction assay and protein analysis.

The role of the recently identified first p53-homologue, p73, in neoplastic transformation is unknown. To elucidate p73 gene expression in hematopoiesis, we investigated samples from chronic myeloid leukemia (CML) and acute myeloid leukemia patients, leukemia cell lines, as well as mature and immature normal hematopoietic cells by real-time quantitative RT-PCR and Western blot analysis. We found a distinct p73 expression profile with highest p73 mRNA transcript levels in hematopoietic malignancies such as CML blast crisis and acute myelogenous leukemia versus CML chronic phase and normal controls. Mono- and biallelic p73 expression was found in both normal and malignant hematopoiesis. p73 protein was expressed at various levels in leukemia samples and cell lines but could not be detected in any normal controls tested. Our results point to a distinct yet undefined role of p73 in the pathogenesis of myeloid neoplasms.