PB1791: ON THE ISSUE OF HOUSEKEEPING GENE PRIMER DESIGN FOR ADEQUATE ASSESSMENT OF THE EXPRESSION OF THE CHIMERIC TRANSCRIPTS.

Abstract

Background: Chimeric transcripts, which are the markers of hematological malignancies, is often assessed by expression level relative to the control housekeeping gene. The critical step for the analysis is RNA isolation and subsequent cDNA synthesis. Routine RNA isolation methods do not guarantee the absence of genomic DNA impurities, which may interfere with cDNA amplification. This results in the bias in control gene expression and underestimation of the relative expression. The use of sophisticated RNA isolation procedures or treatment with RNase-free DNAses significantly complicates and increases the cost of analysis, and can also lead to degradation of target sequences by endogenous nucleases. Here we describe a simple and effective solution to this problem. Aims: To optimize the RT-PCR approach for quantitative assessment of the chimeric transcripts gene expression relative to the control ABL1 gene, for the insensitivity to genomic DNA impurities. Methods: Total RNA was isolated from peripheral blood leukocytes or bone marrow cells as described by Chomczynski (1987). RT-PCR was performed using reagents and primers from Synthol (Moscow, Russia). Results: For ABL1 mRNA amplification primers flanking 2 gene introns were designed (see Table 1). This precludes PCR amplification of the corresponding (139708bp long) genomic sequence. At the same time, the cDNA of this fragment is only 100 nucleotides long. The primers recommended by Europe Against Cancer Association (EAC) and widely used (including as part of commercial diagnostic kits) flank 687bp ABL1 gene DNA fragment. In this case, the admixture of genomic DNA is co-amplified with the cDNA transcript in PCR. We have compared CBFB::MYH11 chimeric transcript relative expression levels in patients with AML, obtained with ABL-EAC primers and those proposed by us (ABL-N). We have tested 40 cDNA samples obtained from diverse RNA isolation routines, including 8 archival samples stored in the freezer for more than 6 months. The threshold cycles of amplification (ΔCt) for the samples from freshly isolated RNA were mostly the same with ABL-EAC and ABL-N primers. For archival RNA, cDNA, and RNA isolated from frozen cells, ΔCt with ABL-N primers drops significantly while ΔCt with ABL-EAC primers is preserved. This may indicate degradation in archival RNA and cDNA samples, while the level of genomic DNA decreases slightly. Thus, usage of ABL-EAC primers may lead in some circumstances to a significant bias in chimeric transcript evaluation. While primers we offer (ABL-N) are still effective even in the cases with compromised sample quality. Image:Summary/Conclusion: Genomic DNA amplification does not tamper with fusion gene transcripts RT-PCR measurement due to the absence of an appropriate target in the genomic DNA. While primers for measuring housekeeping genes expression (even those widely used) may have these targets and therefore amplify genomic DNA along with cDNA. Here we propose primers for measuring ABL1 control gene expression allowing reliable calculation of chimeric transcripts relative expression, regardless of RNA sample purity, in contrast to primers recommended by the EAC.