Potential Pitfalls In Quantitative Real-Time PCR for Molecular Monitoring of BCR-ABL In the Management of CML.
Authors: Zhang Hanfang , Patel Niren , Kutlar Ferdane , Jillella Anand P. , Ustun Celalettin , Wells Leigh G , Clair Betsy , Kutlar Abdullah 
Published: October 2019

Abstract
Abstract 1228
Due to a lack of standardized methodologies and reporting, results of BCR-ABL transcripts by quantitative real-time PCR (qPCR) are difficult to evaluate and compare between laboratories in molecular monitoring of BCR-ABL in CML. Although progress has been made in harmonization of reporting BCR-ABL quantification, such as the introduction of the International Scale, the standardization of each step in qPCR for BCR-ABL quantification is important. The majority of laboratories use an absolute approach with a standard curve to quantitate the transcript level of BCR-ABL. A number of sources are available for standards, such as RNA, cDNA, and plasmid. Among those, plasmid DNA is the most widely used. Since the standard is the most important factor in the absolute approach, in this study different preparations of plasmid DNA were investigated by qPCR with a TaqMan probe to evaluate their amplification rate. BCR-ABL b3a2 and ABL were individually cloned into pGEM-T Easy vector, and each plasmid DNA was isolated with Qiagen Plasmid Maxi kit. After quantification, DNA was diluted to obtain E6 copy number as templates for qPCR. For data comparison, a common threshhold was set in the middle of the exponential phase and Ct values were obtained. A small Ct value indicates a high amplification rate. Multiple preparations of purified completely linearized plasmid DNA for both b3a2 and ABL have the Ct values of 17.90 ± 0.19 and 17.94 ± 0.44 respectively. These values were very similar or equal to the Cts of purified b3a2 and ABL DNA fragments derived from RT-PCR. However, the non-linearized circular plasmid DNA only had 20.36 ± 0.08 for b3a2 and 20.31 ± 0.13 for ABL when the same number of templates was analyzed in the same qPCR indicating a 5.5 fold deficiency in amplification rate. Although the amplification efficiency obtained from both linearized and circular plasmids were very similar in the range of 3.3±0.3, a deficiency in the amplification rate from the circular plasmid indicates that the complicated super coiled structure in the circular plasmid DNA may interfere with the amplification. After plasmid DNA was completely linearized with three individual restriction enzymes, aliquots of known amounts of plasmid DNA were adjusted to E6 copy number and analyzed without further purification. Depending on the restriction enzyme the amplification rate of non-purified plasmid varied from 69 to almost 100% compared with purified linearized plasmid DNA. To mimic the purification procedure, Rnase A was added to the restriction digestion mixture to remove RNA contamination. Without purification, the addition of RNase A significantly inhibits the amplification rate. Furthermore, standards from Ipsogen were compared in the study. From multiple concentration points in comparison of the standards, Ipsogen standards had a Ct value of 1.43 more than the purified completely linearized plasmid DNA with 2.7 fold deficiency in amplification rate. Since the Ct value of the sample is the same, the standards with low amplification rate would over-estimate the copy number of the sample. In theory, the copy ratio of the fusion gene (b3a2) to the control gene (ABL) should be the same, independent on the amplification rate of the standards. However, in practical experiments with a high amplification rate of the standards, the copy ratio is more accurate and reproducible when the samples have a low copy number of BCR-ABL. The combination of incomplete linearization of plasmid DNA in restriction enzyme digestion, non-purification of the linearized plasmid and contamination of interfering substances may together decrease the amplification rate. The preparation of purified completely linearized plasmid provides a standard way to obtain standards for qPCR in molecular monitoring of the BCR-ABL transcript level in the management of CML patients as well as for qPCR in other usages.

Disclosures:
No relevant conflicts of interest to declare.