RT-PCR – Leukaemia Care e-learning

RT-PCR. A key cellular process is the transcription of DNA to RNA by RNA polymerase. RNA introns (non-coding regions) are spliced out and the exons (coding regions) are joined to give messenger RNA (mRNA). mRNA can then be reverse transcribed using a reverse transcriptase to form complementary DNA (cDNA), providing a template for PCR that lacks introns (Figure 2.3).

Exon 1 of ABL1 is joined to exon 13 or 14 of BCR in about 95% of CML cases,allowing use of a standard primer set for 95% of patients with CML.

**Figure 2.3** Standard polymerase chain reaction (PCR) requires different primers for different patients whereas the PCR of complementary DNA (cDNA) can use the same primer sets for most patients. Arrows indicate regions where primers anneal to the DNA.

Quantitative RT-PCR (RT-qPCR). To quantify the BCR–ABL1 cDNA as it is amplified, a fluorescent dye such as Sybr Green is added to the reaction and is incorporated into the DNA. The level of fluorescence (captured by a light detector) is proportional to the amount of BCR–ABL1 cDNA.