Practical considerations – Sample quality

The quality of RT-qPCR detection depends on the quality of the blood or bone marrow sample provided. The red blood cells are lysed and RNA extracted from the remaining cells for RT-qPCR. However, if delivery or processing of the sample is delayed, or the sample has clotted because of prolonged marrow extraction or difficult venepuncture, many of the cells will be dead or dying and will therefore have low levels of poor-quality RNA, reducing transcription. However, transcription of the BCR–ABL1 transcript is reduced proportionally to the ABL1 reference gene, so BCR–ABL1:ABL1 ratios are maintained and RT-qPCR results may still be valid. Nevertheless, the sensitivity threshold for detection may be poorer and it may not be possible to detect the very low levels of BCR–ABL1 needed to identify deep molecular responses.