*Extraction reagent for liquid samples
Recent progress in RNA isolation technology has made it possible to replace lengthy and laborious methods of total RNA isolation (1) by a single-step method (2,3). The RNA STAT-50TM LS 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 liquid samples of human, animal, plant, yeast and bacterial origin. Extensive laboratory tests have shown that RNA STAT-50 TM LS is highly reliable and produces very consistent results.
The composition of RNA STAT-50TM LS includes phenol and Guanidinium thiocyanate in a mono-phase solution. A biological specimen is lysed (or homogenized) in RNA STAT-50TM LS and mixed with chloroform. Following centrifugation, the lysate 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 water. RNA STAT-50 TM LS is the most effective method of RNA isolation from biological fluids such as blood, serum, amniotic fluid, CSF etc. The entire procedure can be completed in 1 hour and the recovery of undegraded mRNAs I s30-150% greater than with any other method of RNA isolation.
The total RNA isolated by RNA STAT-50TM LS is undegraded and free of 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 treatment with DNase or additional purification. The simplicity of the isolation using RNA STAT-50 TM LS makes it possible to process simultaneously a large number of samples, and the excellent recovery of RNA permits the use of this product for isolation of RNA from very small biological specimens.
Reagent supplied: RNA STAT-50TM LS. Reagents required, but not supplied: chloroform, isopropanol and ethanol.
The methods include the
1. Homogenization 0.75 ml RNA STAT-50TM + 0.25 ml sample.
2. RNA Extraction homogenate +0.2 ml chloroform.
3. RNA Precipitation aqueous phase +0.5 ml isopropanol.
4. RNA Wash 1 ml 75% ethanol.
Unless stated otherwise the procedure is carried out at room temperature. The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. Before using test if the tubes can withstand centrifugation at 10,000 x g with the RNA STAT-50TM LS.
A. BIOLOGICAL FLUIDS.
Mix 0.75 ml of RNA STAT-50TM LS with 0.25 ml of sample and lyse cells (or cellular debris) suspended in the sample by passing the mixture several times through a pipette. Use at least 0.75 ml of RNA STAT-50TM LS per 5-10 x 106 cells.
B. TISSUE SUSPENSIONS.
Homogenize 0.25 ml sample with 0.75 ml of RNA STAT-50TM LS in a glass-Teflon or Polytron homogenizer. If the sample vol. is < 0.25 ml, adjust the volume with water. The volume ration of RNA STAT-50TM LS to sample should always be 3 to 1.
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 0.75 ml of RNA STAT-50TM LS, cover the samples tightly, shake vigorously for 15 seconds and let them stay at room temperature for 2-3 minutes, centrifuge the homogenate at 12,000 g (max.) for 15 minutes at 4oC. Following centrifugation, the homogenate separates into two phases: a lower red, phenol-chloroform phase and the colorless upper 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 homogenate.
3. RNA PRECIPITATION
Transfer the aqueous phase to a fresh tube and mix with isopropanol. Add 0.5 ml of isopropanol per 0.75 ml of RNA STAT-50 TM LS for homogenization. Store samples at room temperature for 5-10 min and centrifuge at 12,000 g (max.) for 10 minutes at 4oC. RNA precipitate (often invisible before centrifugation) forms a white pellet at the bottom of the tube.
4. RNA WASH
Remove supernatant and wash the RNA pellet once with 75% ethanol by vortexing and subsequent centrifugation at 7,500 g (max.) for 5 minutes at 4oC. Add at least 1 ml of 75% ethanol per 0.75 ml of RNA STAT-50TM LS used for the initial homogenization. At the end of the procedure, dry briefly the RNA pellet by air-drying or under vacuum (5-10 min). It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Do not use the Speed-Vac for drying. Dissolve the RNA pellet in FORMazolTM (Cat. # T-4025), water or in 0.5% SDS solution. Vortex or pass the pellet a few times through a pipette tip. An incubation for 10-15 minutes at 55-60oC is required to completely dissolve RNA samples. The SDS solution used for RNA solubilization should be made free of RNase by diethyl pyrocarbonate (DEPC) treatment.
NOTE: Should you use our RNase free purified water (Cat. # T1-3200) this treatment step is unnecessary. The final preparation of total RNA is free of DNA and proteins and has a 260/280 ration > 1.8.
NOTES AND COMMENTS
1. Isolation of RNA from small samples (<106 of cells). Lyse (or homogenize) samples in 0.75 ml of RNA STAT-50TM LS in the Eppendorf tube and follow the isolation protocol with the exception of the RNA precipitation which should be carried out for 30 minutes at 4oC. If the expected yield of RNA is <1 ug, an addition of 5 ug of carrier tRNA or glycogen (molecular biology grade, Boehringer Mannheim) to the aqueous phase is necessary for a 100 recovery of the RNA precipitate.
2. Following homogenization (before addition of chloroform) samples can be stored at -70oC for at least two weeks.
3. Hands and dust may be the major source of the RNase contamination. Use gloves and keep tubes closed throughout the procedure.4. Citation of the method: total RNA was isolated by the single-step method (2) using RNA STAT-50TM LS.
SPECIAL HANDLING PRECAUTIONS
The RNA STAT-50TM LS contains poison (phenol) and irritant (guanidinium thiocyanate). CAN BE FATAL. When working with RNA STAT-50TM LS 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.
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, 156-159.
3. Chomzynski P. (1989) The RNAzolTM B method.