Thanks to its precision, sensitivity and flexibility, the qPCR technology is widely used and efficient to explore the gene expression landscape, even for single cell experiments. For robust and successful results in single cell analysis using quantitative real time PCR, crucial steps are necessary: experimental design, sample preparation and single cell collection, direct lysis, Reverse Transcription, Preamplification, qPCR and data analysis. Each step has to be optimized to guarantee sufficient, robust and reliable qPCR results.
The direct lysis step is a fast solution to obtain cell lysates from cell cultures or cell sorting, by avoiding RNA loss, in comparison to standard RNA isolation procedures. Even if this strategy offers a fast and easy-to-use protocol, the formulation of the lysis buffer is however very important to keep high qPCR performance, while protecting RNA from degradation and its adherence to the plastic tubes or wells.
The preamplification is a supplementary and crucial step that allows to perform a specific enrichment of the targeted cDNA sequences, particularly for low expressed genes or for limited sample amount.
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