Cell culture and QRT PCR Gene Expression
Gene expression analysis by QRT-PCR is a powerful and commonly used technique that compares the relative expression of mRNA (reverse transcribed to cDNA) for specific genes, between experimentally treated cells and a non-treated control.
It requires good cell culture and molecular biology methodology to achieve optimal results. Cell lines used should accurately represent the tissue under investigation as much as possible. Cultures should be well maintained, checked for identity, free from contamination and not over-passaged. Cell growth must be carefully scheduled to coincide with experimental timelines. Plan ahead by calculating the number of cells required for experimental treatments, controls and replicate wells, the surface area of the wells and the required seeding density. This will avoid excess use of cell culture reagents and materials whilst ensuring there are enough cells for the experiment. Over-estimate the number of cells required by ~20-30% to provide contingency. Check the treated cells under the microscope during the experiment and note any morphological changes or evidence of toxicity and take photographs.
Set-up additional wells of treated and untreated cells for cell count and viability assessment at time harvest for RNA extraction. Harvest cells quickly for RNA extraction, on ice if possible to halt biological activity. Add RNA extraction homogenisation and stabilisation solutions directly to the cells to prevent RNA degradation. Quantify and qualify extracted RNA and only use high quality, pure RNA for reverse transcription to cDNA. Prepare RT-free controls for all samples and ensure primers and probes are well designed, and if possible, span intron-exon boundaries to ensure amplification is specific to transcribed cDNA.
Check that selected Housekeeping (H-KG) are stably expressed and suitable for the experimental conditions and chosen cell line and consider geNORM analysis to select appropriate H-KGs.
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Written by Jim Cooper, January 2021