Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample
AUTOR(ES)
Marone, Maria
FONTE
Biological Procedures Online
RESUMO
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-β1 in TF-1 cells.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=145543Documentos Relacionados
- Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR
- Serial analysis of gene expression: rapid RT-PCR analysis of unknown SAGE tags.
- Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT–PCR
- A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression
- RT–PCR analysis of 5′ to 3′-end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria