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132 Mokany et al.
2.3. Duplex Single-Tube Real-Time Quantitative RT-PCR
1. BD QTaq DNA polymerase mix (BD Biosciences Clontech, Part No. 639651);
includes BD QTaq DNA polymerase (with built-in, hot start antibody), reaction
buffer, dNTPs, and MgCl 2.
2. Moloney murine leukemia virus (M-MLV) Reverse Transcriptase (RNase H
minus), 200U/µL (Promega Part No. M5301).
3. 50X BD QZyme Gene-Specific Primers (D-tag; 5� and 3� BCR primers) (BD
QZyme Quantitative PCR reagent Part No. 638294, BD Biosciences Clontech;
see Note 2).
4. 50X BD QZyme Gene-Specific Primers (B-tag; 5� and 3� S-type PML-RARα
primers) (BD QZyme Quantitative PCR reagent Part No. 638292, BD Biosciences
Clontech; see Note 2).
5. 50X BD QZyme Gene-Specific Primers (B-tag; 5� and 3� L-type/V-type PML-
RARα primers) (BD QZyme Quantitative PCR reagent, Part No. 638293, BD
Biosciences Clontech; see Note 2).
6. 100X BD QZyme Substrate D (CAL ORANGE).
7. 100X BD QZyme Substrate B (FAM).
8. Calibrators and patient RNA samples as prepared under Subheading 3.
9. MicroAmp ® optical 96-well plate (Applied Biosystems, Part No. N-801–0560).
10. PCR cooler (Eppendorf, cat. no. 3881 000.023). This should be stored in the –
20°C freezer until required.
11. MicroAmp optical caps (Applied Biosystems, Part No. N801–0935).
12. MicroAmp cap installing tool (Applied Biosystems, Part No. N801–0438).
13. ABI PRISM ® 7700 Sequence Detection System and Sequence Detection Software
1.9.1 (Applied Biosystems) and computer (see Note 3).
3. Methods
The single-tube RT-PCR method presented in this chapter displays features
that provide advantages compared to other published real-time methods. The
majority of real-time PCR methods utilize plasmids as calibrators from which
to estimate the number of mRNA transcripts in patient samples (see Note 4).
This method exploits the advantages of both types of calibrators (plasmid and
total RNA) with two calibration curves. Dilutions of the plasmid containing
the full-length fusion target cDNA allow the estimation of copy number (PML-
RARα calibration curve), whereas dilutions of total RNA allow estimation of
endogenous BCR mRNA expression and controls for the activity of the reverse
transcriptase. Both the target and endogenous control mRNA transcripts are
amplified simultaneously from patient samples. The combination of calibrators
chosen in this chapter are designed to mimic the complex milieu of the
patient samples being tested while simultaneously allowing an estimation of
the exact copy number of the target (not achievable from mRNA transcripts in
total RNA) (see Note 4).