Sigma′s GenElute Mammalian Total RNA Miniprep Kit provides a simple and convenient way to isolate total RNA from mammalian cells and tissues. Protocols are provided for cells, tissues, and fibrous tissues. These protocols differ in their cell lysis and disruption conditions. Once the RNA is bound to the GenElute Binding Column, the purification procedure is the same for all starting materials. For fibrous tissues the kit must be used with proteinase K (Cat. No. P4850) to ensure effective cell disruption. The kit combines the advantages of a silica-based system with a microspin format and eliminates the need for cesium chloride gradients, alcohol precipitation, and hazardous organic compounds such as phenol and chloroform. Cells or tissues are lysed and homogenized in a buffer containing guanidine thiocyanate to ensure thorough
denaturation of macromolecules and inactivation of RNases. Addition of ethanol causes RNA to bind when the lysate is spun through a silica membrane in a microcentrifuge tube. After washing to remove contaminants, RNA is eluted in 50-100 μL of Elution Solution. Up to 150 μg of total RNA can be isolated in less than 30 minutes.
If all traces of DNA contamination must be eliminated, further treatment with DNase I is recommended. DNase I digestion can be performed while the RNA is bound to the GenElute Binding Column using the On-Column DNase I Digestion Set (DNASE10 and DNASE70). Alternatively, for more stringent removal of contaminating DNA, the final RNA preparation can be treated with Amplification Grade DNase I (AMPD1).
We are committed to bringing you Greener Alternative Products, which adhere to one or more of The 12 Principles of Greener Chemistry. This product has Inherently Safer Chemistry, compared to the standard use of phenol and chloroform to perform DNA extractions.
GenElute™ Mammalian Total RNA Miniprep Kit has been used to isolate total RNA:
- from snap-frozen preoptic area of rat brain
- from central part of the mantle tissue containing gonads of Mytilus trossulus mussel
- from murine liver
The purified RNA is ready for reverse transcription and PCR, labeling and microarray analysis, and other common applications. Note that RNA shorter than 200 nucleotides in length, such as tRNA, 5S rRNA, and 5.8S rRNA, is not recovered efficiently under the conditions used with this kit.
Features and Benefits
- Purifies total RNA from up to 107 cells or 40 mg of tissue per prep
- Yields up to 150 μg of pure, concentrated total RNA per prep
- Recover RNA from as few as 100 cells
- Simple and efficient–12 to 18 preps in 30 minutes
- Faster than gravity flow anion exchange methods
- No cumbersome steps associated with resins and magnetic slurries
- 40% more purifications per kit than the leading supplier
The GenElute™ Mammalian Total RNA Purification Kit combines silica-membrane technology with a convenient spin column format for a rapid bind, wash, and elute method to prepare high quality total RNA.
For additional information, please see www.sigma-aldrich.com/totalrna.
Samples are lysed and homogenized in guanidine thiocyanate and 2-mercaptoethanol to release RNA and inactivate RNases. Lysates are spun through a filtration column to remove cellular debris and shear DNA. The filtrate is then applied to a high capacity silica column to bind total RNA, followed by washing and elution. Up to 150 μg of total RNA can be recovered per prep in 100 μl of water. The purified RNA is ready for Northern blots (Fig. 1), RT-PCR (Fig. 2) and other common applications.
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