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.
Preparation of Total RNA
Total RNA was prepared from suspension cell line HL60 (5x106, 1.5x107 cells) and adherent cell line HEK293 (6 cm dish, 10 cm dish) following the recommended FastPure protocols. The kit yielded highly purified total RNA, with an A260/A280 ratio exceeding 1.9 for each sample. The RNA quality was further examined by agarose gel electrophoresis (Figure 2).
Total RNA was extracted from 30 mg samples of several mouse tissues (liver, heart, kidney, and brain) obtained from a 10-week old male mouse using the appropriate FastPure protocol. High-purity total RNA (with an A260/A280 ratio exceeding 1.9) was obtained from every sample. Furthermore, this RNA contained no genomic DNA contamination even without DNase treatment (Figure 3).
Total RNA samples from HL60 (5x106 cells) and mouse liver were prepared using the appropriate FastPure protocol (the mouse liver extraction included a DNase treatment). Reverse transcription reactions (10 µL) were performed using 2 pg to 220 ng (10x dilution) of these total RNA samples. A 2 µL aliquot of each reverse transcription reaction was used for templates in real-time PCR (25 µL reaction system, target gene GAPDH) using SYBR® Premix Ex Taq™ and the Thermal Cycler Dice® Real-Time System (both from Takara Bio).
In both cases, highly quantitative real-time RT-PCR results were obtained. qRT-PCR results for the mouse liver total RNA sample showed excellent results with low Ct values and an even distribution of curves over a range of dilutions of the RNA sample (figure not shown). The FastPure RNA Kit enabled preparation of total RNA, with only minimal amounts of genomic DNA contamination without DNase treatment.
Takara Bio, however, recommends the use of an on-column DNase treatment when it is not possible to design PCR primers at an exon junction or when preparing RNA for specific applications in which trace amounts of genomic DNA may cause problems such as analysis of single-copy gene expression.
The FastPure RNA Kit provides a rapid and convenient method for extracting total RNA from cultured cells and mammalian tissues, without the need for organic solvents. The resultant RNA is of high quality and provides excellent results in real-time RT-PCR. For those applications where trace amounts of genomic DNA are a concern, a simple DNase treatment while the RNA is bound to the filter is sufficient to eliminate all DNA contamination.