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Appendix: Method In Focus

Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies

Assay characteristics

The method is based on a commercially available the TaqMan® Pre-Developed Assay Reagents (PDARs) Endogenous Controls using ABI PRISM® 7900HT Sequence Detection System (Table 1 ).

Table 1 TaqMan® Pre-Developed Assay Reagents (PDARs) Endogenous Controls with VIC® dye-MGB probes

Protocol

It is assumed that the user is familiar with the ABI PRISM® 7900HT Sequence Detection System.

RNA preparation

The quality of input RNA is important. RNA can be prepared by several methods; however, in our hands a relatively easy method giving consistent results is RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) using the manufacturer's protocol. Accurate RNA quantification by spectrophotometry is an essential step for standardization of the input RNA amount.

RT reaction

The RNA is reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol with a minor modification, the addition of RNase inhibitor (Applied Biosystems, Foster City, CA, USA) at a final concentration of 1 U/μl. RNA samples (2 μg) are distributed into PCR tubes and nuclease-free water is added to a final volume of 50 μl. Reaction master mix (2 ×) is prepared as follows: Table 2

Table 2

Add 50 μl of the 2X master mix to each RNA sample (final volume 100 μl) and mix gently by pipetting and briefly spining down the content of the reaction. Samples are incubated at 25°C for 10 min and 37°C for 120 min in a PCR machine.

Real-time PCR

PCR reactions is prepared in triplicates at a final volume of 20 μl: Table 3

Table 3

Thermal cycling conditions comprised an initial UNG incubation at 50°C for 2 min, AmpliTaq Gold® DNA Polymerase activation at 95°C for 10 mins, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Each measurement is performed in triplicate. Sequences of primers and probes of the assays are presented in the above table.

Time requirement

Total time: 7–10 h (12–14 h). The time indicated represents the average time requirement for complete processing of a single sample, from RNA extraction (Qiagen) to the final result. The numbers in parentheses indicate the time requirement for processing of 50 samples by a single person.

Costs of the assay

The overall cost of consumables per analysis of one gene expression in a single specimen in triplicate is 6.90$. Laboratory equipment, staff and maintenance are not included in the calculation.

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Correspondence to R Levy.

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Levy, R. Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. Leukemia 17, 796–797 (2003). https://doi.org/10.1038/sj.leu.2402881

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