App Note
RNAGEM Sample Normalization
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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RNAGEM
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.
- Ideal for preparing RNA from mammalian cell culture, laser capture microdissection, FACS-prepared cell populations, cells in suspension, adherent cells, cells stored in RNAlaterTM and cell pellets
- Simplified, hands-off workflow provides RT-PCR and RT-qPCR ready RNA in approximately 5 minutes
- Releases both RNA and DNA with excellent recovery across a wide range of cell numbers
- Requires no further purification for accurate RT-PCR and RT-qPCR analysis
- Inhibitor-free means no harsh chemical washes or multiple steps
- RT-PCR and RT-qPCR reagents can be added directly to the lysate for a simple, streamlined protocol in 96 well plates
- Each kit contains RNAGEM, DNase I, BLUE buffer, 10x DNase buffer, and 10x TE
Learn more about RNAGEM and recent applications. Download our RNAGEM technical overview. Watch our webinar on advances in extracting and stabilizing RNA.