RNA-DNA Stat

RNA-DNA STAT

Key Benefits

*Isolates high quality RNA/DNA from same sample in 90 minutes.
*1ml isolates RNA/DNA from 100 mg tissue or 10 x 106 cells

Pricing

INTRODUCTION
The entire procedure for RNA/DNA isolation using RNA-DNA STAT-60 can be completed in less than 90 minutes. These are the most effective methods for isolation of RNA and DNA isolation from the same sample. The recovery of undegraded RNA and DNA is 30-150% greater than with any other method of RNA/DNA isolation.

APPLICATION
The total RNA/DNA isolated by RNA-DNA STAT-60 is used for Northern and Southern analysis, dot-blot hybridization, poly A+ selection, in vitro translation, RNase protection assay, molecular cloning, and polymerase chain reaction from human, animal, plant without any additional RNase/DNase treatment. Multi-sample processing is a great advantage of using these simple single reagents. Excellent recovery of RNA/DNA permits the use of this product for isolation of RNA/DNA from very small biological samples (biopsies etc.)

PROTOCOL
RNA/mRNA isolation by the RNA-DNA STAT-60 method includes the following steps:
1. Homogenization RNA STAT-60 (1 ml per 50-100 mg tissue, or 5-10 x 10⁶ cells).
2. RNA Extraction 1 vol. of homogenate +0.2 vol. of chloroform
3. RNA Precipitation 0.5 vol. of isopropanol.
4. RNA Wash 75% ethanol. Unless stated otherwise the procedure is carried out at room temperature.

5.1 HOMOGENIZATION
A. Tissues: Homogenized tissue samples in the RNA STAT-60 (1 ml/ 50-100 mg tissue) in a glass-Teflon or Polytron homogenizer. Sample volume should of the RNA STAT-60 used for homogenization.
B. Cells: Cells grown in mono layer are lysed directly in a culture dish by adding the RNA STAT-60 ^TM (1 ml per 5-10 x 10⁶ cells) by repetitive pipetting. Washing cells before addition of the RNA STAT-60^TM should be avoided as this increases the possibility of mRNA degradation.

5.2 RNA EXTRACTION
Following homogenization, store the homogenate for 5 minutes at room temperature 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. Centrifugate the homogenate at 12,000 g (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 inter phase 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 temperature for 5-10 minutes and centrifuge at 12,000 g (max.) for 10 minutes 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 and subsequent centrifugation at 7,500 g (max.) for 5 minutes 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 completely as it will greatly decrease its solubility. Do not use the SpeedVac 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 solutions 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/10⁶ cells): epithelial cells, 10-15 ug, fibrolasts, 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 (1-10 mg): 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 30 minutes 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. Hand 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 PRECAUTIONS
The RNA STAT-60^TM contains poison (phenol) and irritant (guanidinium thiocyante). 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.

DNA WASH
Remove supernatant and wash the DNA pellet once 75% ethanol by vortexing and subsequent centrifugation at 7,500 g (max.) for 5 minutes at 4^oC. Add at least 1 ml of 75% ethanol per 1 ml of the DNA STAT-60 used for the initial homogenization.At the end of the procedure, dry the DNA pellet briefly by air drying or in a vacuum (5-10 min.). Dissolve the DNA pellet in water or in 1 mm EDTA, pH 7. 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 DNA samples.

SPECIAL HANDLING PRECAUTIONS
The DNA STAT-60^TM contains an irritant (guanidinium salts). Can be fatal. When working with DNA STAT-60 use gloves and eye protection (shield, safety goggles). Do not get on skin or clothing. Avoid breathing vapor. Read warning note on bottle.

Additional references available upon request.