Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies

Abstract

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of nonHodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, b-actin, peptidylprolyl isomerase A, b2 microglobulin, protein kinase cGMPdependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, b-glucoronidase and 18S ribosomal RNA). In all, 12 different B-and T-cell lymphoma/leukemia cell lines, 80 B-and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD 260 measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD 260 measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.

Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues

“…There are no housekeeping genes whose expression is constant in all tissues during normal or malignant growth [19,20] . To properly control for the variation in expression of RNAs, endogenous control genes need to be used for each cell type and tumor type in each experimental design [21] . Here, we chose four housekeeping genes with different abundances that have been widely cited in the literature, and exhibit relatively low variation in expression in different lymphoid tissues [9,21] .…”

“…To properly control for the variation in expression of RNAs, endogenous control genes need to be used for each cell type and tumor type in each experimental design [21] . Here, we chose four housekeeping genes with different abundances that have been widely cited in the literature, and exhibit relatively low variation in expression in different lymphoid tissues [9,21] . Our experiments indicated that the GUSB gene exhibited the lowest variation of expression in formalin-fixed, paraffinembedded and frozen lung cancer specimens, and should be used as a suitable endogenous gene to control for RNA quality and quantity.…”

AID is expressed in germinal center B-cell-like and activated B-cell-like diffuse large-cell lymphomas and is not correlated with intraclonal heterogeneity

“…We have also analyzed AID expression in DLBCL expression data reported by Rosenwald et al 20 In addition, AID mRNA expression in Burkitt lymphoma cell lines (Raji and Daudi), GCB-like DLBCL cell lines (SUDHL4, SUDHL6, OCILY7, OCILY19) and ABC-like DLBCL (OCILY10 and OCILY3) cultured with and without 100 U/ml IL-4 (R&D Systems, Minneapolis, MN, USA) was measured by real-time PCR using the Applied Biosystems Assays-on-Demandt Gene Expression Product on an ABI PRISM s 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and was normalized to the 18S expression that was used as an endogenous RNA/cDNA quantity control as we reported previously. 21 …”

PRAME expression in hairy cell leukemia

“…PRAME expression was quantified using the TaqMan system, and intensity was calculated relative to that of PRKG1 gene expression (37). The positive control K562 cell line demonstrated the highest level of relative PRAME expression, 2.1.…”