Real-time pcr assay for quantitation of BCL-6 mRNA

Izidore S. Lossos, Carol D. Jones, James L. Zehnder, Roger Warnke, Ronald Levy

Research output: Contribution to journalArticlepeer-review

Abstract

The BCL-6 proto-oncogene is considered to be involved in the genesis of NHL. Rearrangements due to chromosomal translocations and somatic mutations of the 5' noncoding regulatory region of the BCL-6 gene are potential mechanisms for altering its expression in NHL. However, simple and precise quantitative methods for measurement of the BC1-6 expression are not available. The frequently used immunostaining with antiBCL-6 antibodies is a semi-quantitative method that is difficult to standardize. Therefore, we developed a real-time RT-PCR assay, based on TaqMan technology, for accurate and reproducible determination of BCL-6 mRNA expression. Primers, designed to cross an exon-intron boundary, enabled specific amplification of cDNA and not of genomic DNA. The intra- and the inter-assay coefficient of variability (CV) is 1.7% and 1.9%, respectively. The method was applied to the germinal center (GC) lymphocytes isolated from the tonsils of 3 patients, K562 and HL-60 cell lines that are considered not to express the BCL-6 RNA by conventional methods and tissue specimens obtained form 23 patients with follicle center lymphoma(FCL), 21 patients with primary diffuse large B cell lymphoma (pDLBCL), 6 patients with secondary DLBCL(sDLBCL) due to low grade NHL transformation, 7 patients with CLL-SLL and 6 patients with MCL. BCL-6 RNA levels were referenced to GAPDH expression and calibrated to the BCL-6/GAPDH ratio observed in Raji cell line. The BCL-6 expression in GC lymphocytes was 4.25,11.97 and 21.71. As expected, minimal BCL-6 gene expression was observed in K562 and HL-60 cell lines (<0.12). BCL-6 expression was 8.53±3.87 (Mean ±SD) in FCL, 7.11±2.31 in sDLBCL, 1.8011.49 in pDLBCL, 3.8713.23 in CLL-SLL and 1.39+0.34 in MCL. While the BCL-6 expression was uniformly high in all the FCL and sDLBCL tumor samples, significant variation in the BCL6 expression levels was observed in the other groups. BCL-6 expression was significantly higher in the sDLBCL compared to the pDLBCL group. The latter observation is most probably due to the persistent BCL-6 expression following higher-grade transformation of FCL. Germinal centers or residual follicles were not observed in 5 of the 6 sDLBCL biopsies. The pDLBCL tumors can be divided into two subgroups with high and low BCL-6 mRNA expression. In summary, we have developed a very sensitive, reproducible and robust method for the measurement of the BCL-6 mRNA. This method may have wide investigational applications for the evaluation of the effects of rearrangements and mutations on BCL-6 expression.

Original languageEnglish (US)
Pages (from-to)166b
JournalBlood
Volume96
Issue number11 PART II
StatePublished - Dec 1 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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