RNA-stat 60

Key Benefits

* Total RNA/mRNA in under 60 minutes.
     * Northern blot/PCR* - ready mRNA in under 60 minutes.
     * No further purification required for use in subsequent procedures including Northern blotting and PCR.
* Extracts 30-150% more total RNA/mRNA than any other method.
* Cost effective method requiring less reagent/sample.

Pricing

1. INTRODUCTION
Recent progress in RNA isolation technology has made it possible to replace lengthy and laborious methods of total RNA isolation¹ by a single-step method^2.3 RNA STAT-60^TM is a new and substantially improved version of the single-step method. It is a complete and ready to use reagent for isolation of total RNA from tissues and cells of human, animal, plant, yeast, bacterial, and viral origin. Extensive laboratory tests have shown that the RNA STAT-60 ^TM is highly reliable and produces very consistent results.  The composition of RNA STAT-60^TM (patent pending) includes phenol and Guanidinium thiocyanate in a mono phase solution. A biological sample is homogenized in the RNA STAT-60^TM using a glass-Teflon or Polytron homogenizer. Upon addition of chloroform, the homogenate separates into two phases: aqueous phase and organic phase.  The total RNA remains exclusively in the aqueous phase while DNA and proteins are extracted into an organic phase and interphase.  The total RNA is precipitated from the aqueous phase by addition of isopropanol, washed with ethanol and solubilized in waster.  The entire procedure for RNA isolation using the RNA STAT-60^TM can be completed in 1 hour. This is the most effective method of RNA isolation. The recovery of undegraded mRNAs using the RNA STAT-60^TM is 30-150% greater than with any other method of RNA isolation. RNA STAT-60^TM offers:

2. APPLICATION
    The total RNA isolated by the RNA STAT-60^TM is undegraded and free of protein and DNA contamination. It can be used for Northern analysis, dot blot hybridization, poly A+ selection, in vitro translation, RNase protection assay, molecular cloning, and for polymerase chain reaction (PCR^*) without additional treatment with DNase. The simplicity of the isolation using the RNA
 STAT-60^TM makes it possible to process simultaneously a large number of samples, and the excellent recovery of RNA from very small biological samples     (biopsies, etc.).

3. REAGENTS SUPPLIED
RNA STAT-60TM: 100 ml or 200 ml bottle containing a red solution of RNA STAT-60TM  

PREPARATION: Ready to use
STORAGE: Refrigerate at 2-8oC. Protect from exposure to light.
STABILITY: 9 months. Refer to expiration date stamped on label.
 

Unless stated otherwise the procedure is carried out at room temperature.

5.1 HOMOGENIZATION
    A. TISSUES
         Homogenize tissues samples in the RNA STAT-60^TM (1 ml/50-100mg tissue) in a glass-Teflon or Polytron homogenizer. Sample volume should not  exceed 10% of the volume of the RNA STAT-60^TM used for homogenization.
    B. CELLS
        Cells grown in mono layer are lysed directly in a culture dish by adding the RNA STAT-60TM (1 ml/3.5cm petri dish) and passing the cell lysate several times through a pipette. Cells grown in suspension are sediment then lysed in the RNA STAT-60TM (1 ml per 5-10 x 106 cells) by repetitive pipetting. Washing calls before addition of the RNA STAT-60TM should be avoided as this increases the possibility of mRNA degradation.
 

5.2 RNA EXTRACTION
    Following homogenization, store the homogenate for 5 min at room temp to permit the complete dissociation of nucleoprotein complexes. Next, add 0.2 ml     of chloroform per 1 ml of the RNA STAT-60^TM , cover the sample tightly, shake vigorously for 15 seconds and let it stay at room temperature for 2-3 minutes. Centrifuge the homogenate at 12,000g (max) for 15 minutes at 4^oC.  Following centrifugation, the homogenate separates into two phases: a lower red phenol chloroform phase and the colorless upper aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interferes and organic phase. The volume of the aqueous phase is about 60% of the volume of RNA STAT-60^TM used for homogenization.

5.3 RNA PRECIPITATION
    Transfer the aqueous phase to a fresh tube and mix with isopropanol. Add 0.5 ml of isopropanol per 1 ml of the RNA STAT-60^TM used for homogenization. Store samples at room temp for 5-10 minutes and, centrifuge at 12,000g (max.) for 10 min at 4^oC. RNA precipitate (often visible before centrifugation) forms a white pellet at the bottom of the tube.

5.4 RNA WASH
    Remove supernatant and wash the RNA pellet once with 75% ethanol by     vortexing and subsequent centrifugation at 7,500g (max.) for 5 min at 4^oC. Add at least 1 ml of 75% ethanol per 1 ml of the RNA STAT-60^TM used for the initial homogenization.
    At the end of the procedure, dry the RNA pellet briefly by air-drying or in a vacuum (5-10 min.). It is important not to let the RNA pellet dry completly as it will greatly decrease its solubility. Do not use the Speed-Vac for drying. Dissolve the RNA pellet in water or in 1 mm EDTA, pH 7, or 0.5% SDS solution. Vortex or pass the pellet a few times through a pipette tip. An incubation for 10-15 minutes at 55-60^oC may be required to dissolve RNA samples. Diethylpyrocarbonate (DEPC) treated RNase-free solutions¹ should be used for solubilization of RNA.

6. EXPECTED YIELD AND PURITY
    Expected yield of total RNA:
a.) Tissues (ug/mg tissue): liver, spleen, 7-10 ug; kidney, 3-4 ug; skeletal muscles, brain, 1-1.5 ug; placenta, 1-4 ug.
b.) Cultured cells (ug/10 6 cells): epithelial cells, 10-15 ug, fibroblasts, 5-7 ug. The final preparation of total RNA is free of DNA and proteins and has a 260/280 ratio > 1.8.

7. NOTES AND COMMENTS
    1. For isolation of RNA from a small amount of cells or tissue (1-10mg): homogenize samples in 0.8 ml of the RNA STAT-60^TM, transfer the         homogenate to the eppendorf tube and follow the isolation protocol with the exception of the RNA precipitation which should be carried out for 30m min at 4 ^oC.
    2. Following homogenization (before addition of chloroform) samples can be stored at -70^oC for at least 2 weeks.
    3. An additional precipitation may be necessary to use RNA isolated by the RNA STAT-60^TM in enzymatic assays. Following solubilization, precipitate RNA in the presence of 0.2 M NaCl with two volumes of ethanol for 15 minutes at 4^oC. The PCR and RNase protection assays do not require this         traditional precipitation step.
    4. Hands and dust may be the major source of the RNase contamination. Use gloves and keep tubes closed. The use of sterile, disposable polypropylene tubes is recommended throughout the procedure.
 

8. SPECIAL HANDLING AND PRECAUTIONS
    The RNA STAT-60^TM contains poison (phenol) and irritant (guanidinium thiocyanate). CAN BE FATAL. When working with the RNA STAT-60^TM     use gloves and eye protection (shield, safety goggles). Do not get on skin or clothing. Avoid breathing vapor. Read also the warning note on the bottle. In     case of contact immediately flush eyes or skin with a large amount of water for at least 15 minutes and seek immediate medical attention.

9. REFERENCES
    1. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
    2. Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.  Biochem. 162, 156-189.
    3. Kedzierski, W. and John Porter. 1991. A Novel Non-enzymatic Procedure for Removing DNA Template from RNA Transcription Mixtures.         BioTechniques 10:210-214.

    RNA STAT-60^TM is a trademark of Tel-Test Inc.
    PCR is subject of patents granted to Cetus Corporation.