When working with low cell counts or cell lysates, most rapid methods designed for estimating total RNA quantity (such as optical density or fluorescent dyes) are insufficiently sensitive or affected by material in unpurified lysates.
One solution is to use the DNA in the extracts. qPCR can precisely quantify DNA and this in turn can be used to estimate cell numbers. DNA quantification provides an accurate way to normalize samples – particularly for high-throughput systems using 96 or 384-well plates. To use this approach, DNA must be present in the lysate in a readily amplifiable form and there must be a linear relationship between cell numbers and DNA yield over the expected working range.
RNAGEM is a whole nucleic acid extraction kit that gives linear yields of DNA between <10 cells and ~50,000 cells when using the recommended method. The method can easily be scaled if larger sample sizes are needed.
The amount of RNA in a sample can be normalized using an RT-qPCR. If RNA and DNA are simultaneously co-extracted with similar efficiencies, then gDNA copies can also provide a simple and direct estimate of cell numbers which in turn provides a normalization factor for total RNA quantity.
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Temperature-driven, single-tube extractions of total RNA from mammalian cells, tissues, insects, bacteria and virus
This powerful, broad-specificity approach is ideal for gene expression from a very small number of cells, such as exosomes or single cells, without the need for harsh chemicals or further purification.