Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

Abstract

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-beta1 in TF-1 cells.

Fig. 1. Effect of different MgCl₂ concentrations in the PCR reaction.

Analysis was performed on the human erythroleukemia cell lime TF-1. Bcl-2 amplification was optimal at 1 mM MgCl₂ and decreased gradually at higher concentrations. The intensity of the Aldolase A amplification product did not change significantly. Ald A, Aldolase A.

Fig. 2. Determination of the exponential range of amplification for Bcl-2 and Aldolase A.

The analysis was performed in 1 mM MgCl₂ at 65°C. The intensity of the Bcl-2 amplification product increased up to 36 cycles, whereas Aldolase A(Ald A) reached a plateau at 33 cycles.

Fig. 3. Control for competition between different primer sets.

Reactions were performed in the same conditions for lanes 1, 2 and 3. Lane 1 contained the Aldolase A primer set only, lane 3 the Specific primers for the target RNA, and lane 2 both primer sets. In the selected conditions (1 mM MgCl₂, 65°C, 30 cycles) Bcl-2 and Aldolase A did not compete (left panel). The middle panel shows an example in which the specific primers for the target RNA (p27) competed with the Aldolase A primers (left, 2 mM MgCl₂). The right panel shows the same samples as in the middle panel: substitution of 2 mM with 3 mM MgCl₂, eliminated competition, i.e the intensity of the p27 band did not change when the Aldolase A primers where included in the reaction (lane 2). Ald A, Aldolase A.

Fig. 4. Example of analyses performed on different target RNAs in the same set of samples: effect of TGF-β1 on TF-1 cells.

Lane 1 is control TF-1 cells, lane 2 treatment with TGF-β1, lane 3 treatment with anti-TGF-β1. In all panels, the upper band is the target and the lower band is the amplification product of Aldolase A. T/C indicates the relative level of the target amplification product (T) over the Aldolase A internal control (C) after normalization to the control sample, i.e. it expresses the change in folds with respect to the untreated control (lane 1).