Real-time PCR (qPCR) analysis has become the method of choice for quantitating DNA in a wide variety of applications. The availability of commercial kits has made the technique easy to perform, efficient, and reliable. qPCR methods are easily adapted to high-throughput assays, allowing researchers to process large numbers of samples in a short period of time.
In particular, real-time RT-PCR (qRT-PCR) is a popular technique for determining the relative levels of gene expression among different tissues or species. For efficient qRT-PCR analysis, it is critical to reliably obtain highly purified total RNA from samples easily and quickly.
Conventional methods of total RNA purification have relied on variations of the guanidinium thiocyanate method. In this technique, the RNA is extracted from cells or tissues following treatment with acidified phenol/chloroform and isopropanol precipitation. This method is time consuming and involves the use of organic solvents that are hazardous and may require special procedures for disposal.
In contrast, the FastPure™ RNA Kit from Takara Bio (www.takarabiousa.com) uses a specially designed polymer filter for immobilization of nucleic acids and does not require organic solvents. Because of the filter membrane’s large surface area, specific binding properties, and uniform porosity, total RNA can be extracted in high yield from cultured cells or mammalian tissues.
The filter used in this kit is also exceptionally thin in comparison to widely used glass-fiber filters. Each extraction in the kit is designed to process cells cultured on a 6 or 10 cm diameter dish, 3x106 to 1.5x107 cells grown in suspension, or 5–30 mg of mammalian tissue samples. The resulting purified total RNA is suitable for use in a variety of applications including RT-PCR, Northern blotting, and microarray analysis.
The first step of this protocol is preparation of a lysate by homogenization using the provided lysis and solubilization buffers. Next, total RNA is collected by centrifugation of the lysate through a cartridge containing the polymer filter. The filter is then washed several times and the RNA eluted with the elution buffer.
In contrast to conventional spin-column extraction kits, the FastPure RNA Kit produces highly pure total RNA with minimal genomic DNA contamination when samples are processed without DNase treatment. If desired, DNase treatment can be performed while the RNA is bound to the filter to remove even trace amounts of genomic DNA.
The FastPure RNA Kit provides several protocols, depending on the type of sample and quantity to be used. Figure 1 illustrates the procedure for RNA extraction from 5–15 mg of animal tissue. The Table shows an estimate of the expected total RNA yield from various mouse tissues and cultured cells using this kit.