Myeloid Leukemia

Duplexed QZyme RT-PCR for APL Analysis 131
Fig. 1. Strategy for duplex single-tube reverse-transcription polymerase chain reaction
(PCR) for PML-RARα and BCR. Sequential reactions in single-tube duplex reaction
are: (1) reverse transcription of each transcript with 3� primer making cDNA
copy, inactivation of reverse transcription, and activation of polymerase; (2) PCR with
5� QZyme primer and 3� primer to generate amplicons containing active
DNAzymes; (3) real-time quantitation of transcripts.
3. The human cell line Meg-01 (DSMZ No ACC 364) was expanded in culture
according to conditions recommended by the supplier. Total RNA from this cell
line was used as a negative control for PML-RARα transcripts and construction
of calibrators.
4. Nuclease-free water (Ambion, Inc., Part No. AM-9930) is used at all stages of
this protocol to prevent RNA degradation.
2.2. Extraction and Quantification of Total RNA
1. TRIzol reagents (Life Technologies, Part No. 15596–026).
2. QIAamp RNA Blood Mini Kit (50) (Qiagen, Part No. 52304).
3. 10X MULTI-CORE Buffer (Promega, Part No. R9991).
4. RQ1 RNase-Free DNase (Promega, Part No. M6101, 1 U/µL).
5. RNasin® Ribonuclease Inhibitor (Recombinant), 40 U/µL (Promega, Part No.
N2515).
6. MgCl 2, 25 mM (Applied Biosystems, Part No. N808–0130).