I. Description
III. Creating a Ribonuclease-Free Environment
IV. RNA Isolation Procedure
A. Preparations
B. Tissue Disruption
C. RNA Extraction
D. RNA Precipitation
E. RNA Wash
VI. Determination of RNA Yield and Quality
A. Yield
B. Purity
C. Integrity
VII. Composition of Buffers and Solutions
VIII. Related Products
IX. References
The purity and integrity of isolated RNA is critical for its effective use in procedures such as Northern blotting, oligo(dT) selection of polyA(+) RNA, cDNA synthesis, and translation in vitro. This is especially true for the construction of cDNA libraries where efficient cDNA synthesis requires highly purified mRNA.
The successful isolation of intact RNA by any procedure requires that four important steps be performed: 1) effective disruption of cells or tissue, 2) denaturation of nucleoprotein complexes, 3) inactivation of endogenous ribonuclease (RNase) activity, and 4) purification of RNA away from contaminating DNA and protein. The most important of these is the immediate inactivation of endogenous RNase activity which is released from membrane bound organelles upon cell disruption. The RNAgents® Total RNA Isolation System utilizes two of the most potent known inhibitors of RNase, guanidine thiocyanate and ß -mercaptoethanol (1). In addition, all procedures are done on ice, which significantly slows the rate of RNA degradation (2). Guanidine Thiocyanate, in association with N-lauroyl sarcosine, also acts to disrupt nucleoprotein complexes, allowing RNA to be released into solution and isolated free of protein. Intact RNA is purified away from contaminants by phenol:chloroform extraction (3) based on the rapid one-step procedure of Chomczynski and Sacchi (4). RNA selectively partitions into the aqueous phase, free from DNA and protein, and is easily concentrated by precipitation with Isopropanol. The procedure is easy to perform with large or small quantities of tissue or cultured cells and can be used to process multiple samples in as little as 3 hours. The use of phenol:chloroform extraction eliminates the need for lengthy selective ethanol precipitation steps, overnight ultracentrifugation through cesium chloride gradients, and the use of lithium chloride (LiCl) precipitations. LiCl precipitations can result in the loss of RNAs smaller than 5.8S (5) and carryover of lithium salts can inhibit subsequent cDNA synthesis reactions (6).
RNA purified with the RNAgents® System can be readily used for oligo(dT) selection of polyA(+) RNA, Northern blots, cDNA synthesis and translation in vitro.
Product Size Cat.# --------------------------------------------------------------------------------- RNAgents® Total RNA Isolation System 6 isolations Z5110
This system contains sufficient reagents for six RNA isolations, each from 1 gram of tissue or 1 x 10^8 cultured cells. Includes:
Storage: All of the components may be stored at room temperature. However, storage of Phenol:Chloroform:Isoamyl Alcohol at 4°C is recommended for increased stability. For convenience and to extend the shelf life of the system, Guanidine Thiocyanate is supplied in two preweighed bottles containing 25g each. This allows the denaturing solution to be prepared freshly on two separate occasions simply by adding buffer.
Stability: All components are stable for six months from
the date of purchase. The denaturing solution is stable for up
to three months from the date of preparation when stored at 4°C.
Note: N-lauroyl sarcosine may precipitate out of solution.
If this occurs, heat to redissolve before using.
Caution: Guanidine Thiocyanate is a potent chaotropic agent and irritant. In addition, phenol is poisonous and can cause severe burns. Proper laboratory clothing including gloves and goggles should be worn when handling these reagents. If phenol should contact your skin, rinse the area immediately with large quantities of water and seek medical attention. DO NOT RINSE WITH ETHANOL! For more complete precautions, refer to the appropriate material safety data sheet (MSDS) supplied with the System.
Ribonuclease is difficult to inactivate. Therefore, it is of utmost importance that care be taken to avoid inadvertently introducing RNase activity into your RNA during or after the isolation procedure. This is especially important if the starting material has been difficult to obtain. The following notes will help you to prevent accidental contamination of your sample.
For cultured cells:
To obtain optimal performance from this system, the samples to be extracted should be as fresh as possible. However, if it is not possible to isolate RNA immediately after obtaining the sample of interest, the sample may be frozen with liquid nitrogen and stored at -70°C for future use. The procedures described below are intended for isolating RNA from 1 gram of tissue or 1 x 10^8 cultured cells. The volumes of reagents may be adjusted proportionally for different amounts of starting material.
1. Cells Grown in Suspension Culture
Collect 1 x 10^8 cells in a sterile 50ml conical centrifuge tube by centrifugation at 300 x g for 5 minutes at 4°C. Wash the cell pellet with 25ml of ice-cold, sterile 1X PBS and centrifuge as above to collect the cells. Pour off the supernatant, add 15ml of prechilled denaturing solution, and homogenize as described for animal tissue, Step B.4.b. Then, proceed to Step C.1, below.
2. Cultured Cells Grown in a Monolayer
Different cell lines will grow to different cell densities. Calculate the total number of flasks needed to provide approximately 1 x 10^8 cells. Pour off the culture media from each flask and wash the cells with ice cold, sterile 1X PBS.
a. Add 8ml of prechilled denaturing solution to one of the flasks and rotate it over the cells until you see them lyse. The solution will become very viscous.
b. Transfer the solution from flask #1 to flask #2 using a sterile 10ml glass pipet, lyse the cells, transfer the solution to flask #3, lyse the cells. Transfer the denaturing solution to each flask to be used for the isolation.
c. Add 4ml of denaturing solution to flask #1. Rotate the solution thoroughly over the bottom of the flask to remove any remaining cells and then transfer this solution successively to the other flasks. Note: If you feel that all of the cells have not been removed, use an additional 5-10ml of denaturing solution to wash the flasks. In this case, however, you will have to proportionately increase the amounts of other reagents to be added subsequently .
d. Transfer the 12ml of lysed cells from the last flask to a sterile 50ml centrifuge tube and homogenize the cells as described in Step B.4.b. Then proceed to Step C.1, below.
3. Plant Tissue
a. Dispense 12ml of denaturing solution into a sterile 50ml cell culture tube and chill on ice for 5 minutes.
b. Freeze 1g of freshly harvested tissue in liquid nitrogen.
c. Grind the tissue under liquid nitrogen using a ceramic mortar and pestle.
d. Allow the liquid nitrogen to evaporate, transfer the ground plant tissue to a sterile 50ml conical cell culture tube, and homogenize as described in Step B.4.b. Then proceed to Step C.1, below.
4. Animal Tissue
a. Dispense 12ml of denaturing solution into a sterile 50ml cell culture tube and chill on ice for 5 minutes.
b. Place 1 gram of tissue (either fresh or frozen) into the denaturing solution and disrupt the tissue with a high speed homogenizer such as the Brinkmann Polytron set on high for 15-30 seconds. This usually can be performed in a sterile 50ml conical cell culture tube. Alternatively, mince the tissue and disrupt with a Dounce glass-Teflon homogenizer. Then proceed to Step C.1, below.
PROBLEM: Low A260/A280 ratios
Typically due to protein contamination.
Several methods may be used for further removal of contaminating protein from RNA. The most expedient method is to perform an additional phenol:chloroform extraction on the purified RNA. Repeat Steps C.1 through D.2, above, adding a volume of Phenol:Chloroform:Isoamyl Alcohol equal to that of the final resuspended RNA pellet. This procedure should yield higher A260/A280 ratios. However, some loss of RNA (up to 40%) may be expected.
PROBLEM: Low A260/A230 ratios
Typically due to Guanidine Thiocyanate contamination.
Precipitate the RNA by adding 0.1 volume of 2M Sodium Acetate, pH 4.0, and an equal volume of Isopropanol. Incubate at -20°C for 30 minutes and collect the RNA by centrifugation at 10,000 x g for 15 minutes at 4°C. Resuspend the RNA in 1mM EDTA (RNase-free) and precipitate the RNA as described above. Wash the final pellet with 10ml of 75% ethanol, dry the pellet in a vacuum desiccator for 15-20 minutes, and resuspend in RNase-Free Water.
PROBLEM: RNA degradation
RNase introduced by handling, or RNase not entirely inactivated.
One of the common misconceptions about RNA isolation is that RNase is irreversibly denatured with Guanidine Thiocyanate salts. If a small amount of denatured RNase is present at the end of the procedure, it may renature once the denaturing agents are removed and then degrade the sample. This is most likely to occur with samples that contain very high amounts of endogenous RNase, such as rat pancreas. If this is a problem, use the following procedure, which is based on the isolation protocol of Han et al(2) to eliminate such carryover RNase activity in subsequent RNA isolations.
The RNAgents® Total RNA Isolation System yields high amounts of intact RNA from a variety of cell sources. The yield of total RNA obtained may be determined spectrophotometrically at 260nm, where 1 A260 unit = 40µg of single-stranded RNA/ml. Table 1 lists representative yields of total RNA isolated from a variety of tissue sources using the RNAgents® System.
Table 1. Yields of Total RNA from a Variety of Tissues
RNAgents®
Tissue Total RNA Yield
----------------------------------------------------
HeLa cells 1.6mg RNA/10^8 cells
Mouse intestine 2.3mg RNA/g tissue
Mouse spleen 8.3mg RNA/g tissue
Mouse lung 1.9mg RNA/g tissue
Mouse kidney 3.1mg RNA/g tissue
Mouse liver 8.1mg RNA/g tissue
RNA properly isolated with the RNAgents® System is substantially free of DNA and contaminating protein and may be used directly for oligo(dT) selection of poly A(+) RNA, Northern blots, cDNA synthesis, and translation in vitro. Pure RNA will exhibit A260/A280 ratios of 2.0. However it should be noted that, due to the variations between different starting materials and individual variation in performing the procedure, you should expect to obtain RNA having A260/A280 ratios ranging from 1.7-2.0. If the RNA exhibits a ratio lower than this, refer to Section V, above, for ways to further purify the RNA.
The isolated RNA should also exhibit an A260/A230 ratio greater than 2.0. A ratio lower than this is generally indicative of contamination with Guanidine Thiocyanate that was carried over during the precipitation steps. See Section V, above.
Determine the integrity of the purified RNA by denaturing agarose gel electrophoresis. Several methods are suitable for this purpose, utilizing either formaldehyde (7), glyoxal (8) or methyl mercury hydroxide (9) . When resolved by one of these electrophoretic methods, the 28S and 18S eukaryotic ribosomal RNAs should exhibit a near 2:1 ratio of ethidium bromide staining, indicating that no gross degradation of RNA has occurred. In RNA samples that have been degraded, this ratio will be reversed since the 28S ribosomal RNA is characteristically degraded to an 18S-like species. Refer to Sections III and V, above, for ways to avoid RNA degradation.
10X PBS buffer (per liter):
11.5g - Na
2
HPO
4
2g - KH
2
PO
4
80g - NaCl
2g - KCl
Dissolve in 1 liter of sterile, deionized water. The pH of 1X
PBS will be 7.4.
CSB Buffer:
42mM - sodium citrate, pH 4.0
0.83% - N-lauroyl sarcosine
0.2mM -
ß
-mercaptoethanol
Product Cat.# --------------------------------------------------------------------------------- PolyATtract® mRNA Isolation System I Z5210
This system contains sufficient reagents (excluding the Magnetic Separation Stand) for three separate mRNA isolations, each from 1-5mg total RNA.
Product Cat.# --------------------------------------------------------------------------------- PolyATtract® mRNA Isolation System II Z5200
This system contains sufficient reagents for three separate mRNA isolations, each from 1-5mg total RNA.
Product Cat.# --------------------------------------------------------------------------------- PolyATtract® mRNA Isolation System III Z5300
This system contains sufficient reagents for fifteen separate mRNA isolations, each from 100µg-1,000µg total RNA.
Product Cat.# --------------------------------------------------------------------------------- PolyATtract® mRNA Isolation System IV Z5310
This system contains sufficient reagents (excluding the Magnetic Separation Stand) for fifteen separate mRNA isolations, each from 100µg-1,000µg total RNA.
Product Cat.# --------------------------------------------------------------------------------- PolyATtract® System 1000 Z5420
This system contains sufficient reagents to isolate mRNA from up to 2 grams of tissue or for 4 isolations from 10^8 tissue culture cells.
Product Cat.# --------------------------------------------------------------------------------- PolyATtract® System 1000 Z5400
This system contains sufficient reagents (excluding the PolyATtract® System 1000 Magnetic Separation Stand) to isolate mRNA from up to 2 grams of tissue or for 4 isolations from 10^8 tissue culture cells.
Product Size Cat.#
---------------------------------------------------------------------------------
PolyATtract® Series 9600™ mRNA Isolation System
with cDNA Synthesis Reagents 3- 96 sample Z3790
isolations
Contains all the components necessary to isolate mRNA and synthesize cDNA from three sets of 96 samples. The PolyATtract® Series 9600™ Multi-Magnet is required, but must be purchased separately.
Product Size Cat.#
---------------------------------------------------------------------------------
PolyATtract® Series 9600™ mRNA Isolation System
without cDNA Synthesis Reagents 3- 96 sample Z3890
isolations
Contains all the components necessary to isolate mRNA from three sets of 96 samples. The PolyATtract® Series 9600™ Multi-Magnet is required, but must be purchased separately.
Product Size Cat.#
---------------------------------------------------------------------------------
MagneSphere® Technology
Magnetic Separation Stand (two-hole) 0.5ml Z5331
1.5ml Z5332
12 x 75mm Z5333
---------------------------------------------------------------------------------
MagneSphere® Technology
Magnetic Separation Stand (twelve-hole) 0.5ml Z5341
1.5ml Z5342
12 x 75mm Z5343
---------------------------------------------------------------------------------
PolyATtract® System 1000 Magnetic Separation Stand Z5410
PolyATtract® Series 9600™ Multi-Magnet Z3811
1. Chirgwin, J.M. et al. (1979) Biochemistry 18 , 5294.
2. Han, J.H. et al. (1987) Biochemistry 26, 1667.
3. Perry, R.P. et al. (1972) Biochem. Biophys. Acta 262, 220.
4. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.
5. Stern, D.B. and Newton, J. (1986) Meth. Enzymol. 118 , 488.
6. Wallace, D.M. (1987) Meth. Enzymol.152, 41.
7. Lebrach, H.D. et al. (1977) Biochemistry 16 , 4743.
8. McMaster, G.K. and Carmichael, I. (1977) Proc. Natl. Acad. Sci. USA 74, 4135.
9. Bailey, J.M. and Davidson, N. (1976) Anal. Biochem. 195, 393.
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