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Single Reagent For RNA Isolation
- Single-tube disruption, separation procedure
- High quality intact RNA from tissue and cells
- Ready-to-use reagent and simple procedure saves time
- Entire procedure can be performed in 1 hr
RNAwiz™ is a single homogeneous reagent for the isolation of total RNA from animals, plants and microorganisms. It contains a unique combination of denaturants and RNase inhibitors which are effective even with tissues rich in polysaccharides, fatty acids or proteins. Since the proteins partition into the organic phase, no protease digestion is required. The ease and speed of the procedure allows researchers to process a large number of samples simultaneously. Quick Isolation of RNA The protocol is simple. First, the tissue or cells are homogenized in RNAwiz (see "Storing Tissue Lysates for Future Use" below). The homogenate separates into 3 phases when mixed with chloroform and centrifuged. The upper aqueous phase contains the RNA, the lower organic phase contains the proteins and cellular debris, and the DNA collects at the interphase as a semi-solid. The RNA is precipitated from the aqueous phase with isopropanol. The entire RNA isolation procedure can be completed in about 1 hour, starting with fresh, frozen, or RNAlater™-treated tissue or cells. The isolated RNA is intact, free of most DNA and proteins, and can be used for RT, RT-PCR, ribonuclease protection assays, Northerns, mRNA isolation and in vitro translation (DNase I treatment is recommended prior to RT-PCR). Storing Tissue Lysates for Future Use Several customers have inquired about the long-term storage of tissue and cell homogenates in RNAwiz™. This is useful for researchers carrying out experiments involving time points, and for those who wish to process multiple samples collected over a few days, weeks or months. Experiments carried out at Ambion indicate that tissues homogenized in RNAwiz can be stored at 4°C for several weeks, and at -20°C or -80°C for several months (Figure 1). | | | Figure 1. Stability of Tissue Lysates. A homogenate of mouse spleens in RNAwiz™ was prepared, and aliquots stored at room temperature (r.t.), 4°C, -20°C and -80°C. Total RNA was isolated from the homogenates after various incubation periods. The RNA was electrophoresed in a denaturing gel and transferred to positively charged nylon membranes. The membranes were hybridized with a high specific activity mouse GAPDH RNA probe overnight at 68°C, and exposed to film with an intensifying screen for 6 hours at -80°C. Ambion's NorthernMax™ Kit was used for the electrophoresis, transfer and hybridization. | |