TY - JOUR
T1 - Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies
AU - Lossos, I. S.
AU - Czerwinski, D. K.
AU - Wechser, M. A.
AU - Levy, R.
N1 - Funding Information:
This work was supported by Grants CA33399 and CA34233 from the USPHS-NIH.
PY - 2003/4/1
Y1 - 2003/4/1
N2 - 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 non-Hodgkin'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, β-actin, peptidylprolyl isomerase A, β2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, β-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 OD260 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 OD260 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.
AB - 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 non-Hodgkin'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, β-actin, peptidylprolyl isomerase A, β2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, β-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 OD260 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 OD260 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.
KW - Endogenous controls
KW - Lymphoma
KW - Real-time quantitative RT-PCR
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U2 - 10.1038/sj.leu.2402880
DO - 10.1038/sj.leu.2402880
M3 - Article
C2 - 12682639
AN - SCOPUS:0037397619
VL - 17
SP - 789
EP - 795
JO - Leukemia
JF - Leukemia
SN - 0887-6924
IS - 4
ER -
