Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample.
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.
Department of Gynecology and Department of Hematology. Catholic University, L.go A. Gemelli 8, 00168 Rome. Italy. maria.marone@tiscalinet.it
Maria
M
Marone
Simona
S
Mozzetti
Daniela
D
De Ritis
Luca
L
Pierelli
Giovanni
G
Scambia