Low pH RNA isolation reagents, method, and kit

The present invention describes an RNA isolation process which utilizes low pH reagents. In addition, the reagents are less hazardous and are more stable than those used in prior art methods. This rapid method may be used to obtain purified RNA from a variety of biological sources including human whole blood, plant and animal tissues, cultured cells, body fluids, yeast, and bacteria.

BACKGROUND OF THE INVENTION 
Ribonucleic acid (RNA) purified from biological material is utilized 
extensively for molecular biology research and is becoming an important 
tool in human clinical testing. Most commonly, the isolated RNA is 
characterized by size and quantity to provide diagnostic information about 
both normal and aberrant functioning of genes. For example, gross DNA 
rearrangements associated with common leukemias are detected by isolation 
and identification of abnormal, hybrid RNAs. 
Typically, there are three aspects of isolating substantially undegraded 
RNA from biological samples: (1) the cells or viral protein coats are 
lysed to release RNA; (2) ribonucleases (RNases) are inactivated to 
prevent RNA degradation; and (3) contaminants are removed to purify the 
preparation. Because of the abundance and stability of RNases in 
biological materials, it is important that cell or protein coat lysis and 
RNase inactivation be substantially simultaneous. Therefore, in its 
simplest form, the isolation of RNA is reduced to just two main steps: (1) 
cell lysis (or protein denaturation)/RNase inactivation; and (2) RNA 
purification. 
Several lysing reagents have been formulated to lyse cells and/or viral 
protein coats and inactivate RNases substantially simultaneously. A lysate 
is created by mixing suspended cells (or biological fluid) with the lysing 
reagent, or by grinding tissues with a pestle in the presence of the 
lysing reagent, which facilitates penetration of the lysing reagent. The 
lysate reagent typically contains a detergent to dissolve cells and to 
solubilize proteins and lipids. A strong protein denaturant (i.e., 
denaturing agent) is usually added to aid in inactivating RNases. In 
addition, a strong reductant is often included to ensure complete protein 
denaturation. 
The most common detergents used in lysing reagent formulations are the 
anionic detergents sodium dodecyl sulfate (SDS) and N-lauroyl sarcosine as 
described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd 
ed., 7.3-7.24, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) 
and Ausubel, et al., Current Protocols in Molecular Biology., 4.0.4-4.5.3 
and 13.12.1-13.12.3, John Wiley & Sons, New York (1989). Also, nonionic 
and cationic detergents have been described for this purpose by Favaloro 
et al., Methods Enzymol., 65, 718-749 (1980) and Macfarlane, (U.S. Pat. 
No. 5,010,183), respectively. Typically, nonionic detergents are 
undesirable because they are generally ineffective at inactivating RNases 
in tissues with high nuclease activity. Cationic detergents are generally 
undesirable because they are more hazardous than nonionic and anionic 
detergents. For example, the rat intravenous LD50 is 1200 mg/kg for the 
nonionic detergent Triton X-100 and 118 mg/kg for the anionic detergent 
SDS, but only 6.8 mg/kg for the cationic detergent 
dodecyltrimethylammonium bromide. 
Strong protein denaturants are commonly added to the lysing reagent to 
ensure inactivation of RNases. The most effective and widely used is 
guanidinium thiocyanate, which is described by Ullrich et al., Science, 
196, 1313-1319 (1977) and Chirgwin et al., Biochemistry, 19, 5294-5299 
(1979). Less commonly used as RNase inhibitors are organoclays, which are 
described by Ness et al. (U.S. Pat. No. 5,393,672). 
Other denaturing agents that have been used are guanidine hydrochloride and 
urea, which are described by Cox in Methods Enzymol., 12, 120-129(1968) 
and Auffray et al., Eur. J. Biochem., 107, 303-314(1980), respectively. 
These denaturing agents, however, are less effective at inactivating 
RNases than guanidinium thiocyanate. The addition of a proteolytic enzyme, 
such as Proteinase K, to digest RNases is another strategy used in RNA 
isolation techniques. This also is less effective than guanidinium 
thiocyanate because it is generally too slow at inactivating RNases 
causing RNA degradation, particularly in solid tissue preparations. 
In addition to this primary denaturant, it is common practice to add a 
second denaturant, such as the sulfhydryl reducing agent 
2-mercaptoethanol, to the lysing reagent to ensure complete protein 
denaturation. This denaturant is highly toxic and has a pungent odor, and 
is therefore not easy to use. Furthermore, it is also subject to oxidative 
degradation and therefore reduces the shelf-life of lysing reagents. 
An important factor to consider in the formulation of lysing reagents is 
pH. It has been shown by Noonberg et al., BioTechniques, 19, 731-733 
(1995) that for lysing reagents containing organic solvents, the lower the 
pH, the lower the degree of RNA degradation, within the pH range of 5.5 to 
8.0. However, a review of RNA isolation methods indicates that the pH of 
the lysing reagent is no lower than 4.0 (Chomczynski, U.S. Pat. No. 
4,843,155), and can be as high as 9.0 (Bugos et al., BioTechniques 19, 
734-737 (1995), with most in the neutral range of 7.0-8.0. Chomczynski 
teaches, however, that a pH of lower than 4 results in a significantly 
lower degree of RNA isolation. 
After cell or protein coat lysis and RNase inactivation, RNA is purified by 
isolating it from the complex lysate. There are two general strategies in 
widespread use for liquid phase purification of RNA. These are 
differential centrifugation and solvent extraction combined with salt 
precipitation. 
To separate RNA from deoxyribonucleic acid (DNA) and protein contaminants 
using differential centrifugation, typically the lysate is placed onto a 
solution of cesium chloride as described by Glisin et al., Biochem., 13, 
2633-2637 (1974) and Chirgwin et al., Biochemistry, 19, 5294-5299 (1979). 
Then the sample is centrifuged at high speed (at least 130,000.times. g) 
for at least 12 hours to selectively sediment the RNA, leaving 
contaminants in the supernatant fraction. This method has the 
disadvantages of being very time-consuming, requiring the use of expensive 
ultracentrifugation equipment, and it does not efficiently recover low 
molecular weight RNAs, such as 5S ribosomal RNAs and transfer RNAs. 
The second strategy for RNA purification is to mix the lysate with both an 
organic solvent (typically, phenol and chloroform) and a salt (typically, 
sodium acetate). Phenol not only denatures proteins but, following 
centrifugation, causes the protein to collect at the interface between the 
organic and aqueous layers. Chloroform facilitates the separation of 
organic and aqueous phases. Such phenol-based reagents, however, are 
typically unstable during storage due to oxidation. 
At low pH (e.g., 4-7), the addition of a high concentration (e.g., 2-3 
molar) salt solution causes DNA to selectively precipitate so that 
following centrifugation, it too will collect at the organic-aqueous 
interface. Thus, by combining the phenol extraction with salt 
precipitation, both proteins and DNA collect at the interface following 
centrifugation, leaving RNA in the supernatant. This is described, for 
example, by Chomczynski et al., Anal. Biochem., 162 156-159 (1987) and 
Chomczynski, EP 0 554 034. 
The salt solutions generally used in solvent extraction-salt precipitation 
techniques are typically sodium acetate solutions of pH 4.0 to pH 7.0 at 
concentrations of 2-3 molar. An alternative salt, lithium chloride, 
selectively precipitate RNA rather than the contaminating DNA. The 
addition of this salt to the aqueous fraction, recovered after 
phenol-chloroform extraction, is described by Ausubel et al., Current 
Protocols in Molecular Biology, 4.0.4-4.5.3. John Wiley & Sons, New York 
(1989) and Auffray et al., Eur. J. Biochem., 107, 303-314 (1980). However, 
a disadvantage of lithium chloride precipitation is that the low molecular 
weight RNAs are not recovered. 
Reagents required for isolating RNA in conventional methods are formulated 
typically using organic solvents and other generally hazardous chemicals. 
For example, the raw materials in wide use are listed below, along with 
label precautions and toxicity information as obtained from Sigma Chemical 
Company. The toxicity data are given as LD50 values where the lower the 
LD50 value, the more hazardous the compound. Generally, lysing and/or 
purification solutions contain: chloroform, which is highly toxic and may 
cause cancer, having an LD50 of 908 mg/kg (rat oral administration); 
guanidinium thiocyanate, which is considered harmful, having an LD50 of 
300 mg/kg (mouse intraperitoneal injection); 2-mercaptoethanol, which is 
considered highly toxic and has a very strong odor stench, having an LD50 
of 244 mg/kg (rat oral administration); and phenol, which is highly toxic, 
having an LD50 of 317 mg/kg (mouse oral administration). 
A method for DNA and RNA isolation that uses less hazardous compounds, such 
as benzyl alcohol to replace phenol and chloroform, is disclosed by Ness 
et al., U.S. Pat. No.5,393,672. Despite the lower toxicity of benzyl 
alcohol, it is still classified as harmful with an LD50 of 1230 mg/kg by 
rat oral administration. In addition, even less toxic organic solvents 
require special handling and disposal. 
Thus, there is a need in the field for a method that is less hazardous 
and/or does not involves the use of organic solvents. In addition, there 
is a need for reagents that are more stable at room temperature (i.e., 
20-30.degree. C.). Also, there is a need for relatively rapid protocols to 
isolate RNA from a variety of biological materials, especially for routine 
testing as found in clinical laboratories. 
SUMMARY OF THE INVENTION 
The present invention provides a kit for isolating RNA comprising 
instruction means for isolating substantially undegraded RNA from a 
biological sample and a Cell Lysis Reagent. The Cell Lysis Reagent 
includes: an amount of an anionic detergent effective to lyse cells or 
protein coats sufficiently to release substantially undegraded RNA; a 
chelating agent; water; and an amount of a buffer effective to provide a 
pH of less than about 6 (preferably, less than about 5, and more 
preferably, less than about 4). The anionic detergent is preferably a 
dodecyl sulfate salt or N-lauroyl sarcosine. The chelating agent is 
preferably EDTA or CDTA. 
In addition to the Cell Lysis Reagent, the kit can include a Protein-DNA 
Precipitation Reagent comprising a sodium or potassium salt in an amount 
effective to precipitate DNA and protein, water, and an amount of a buffer 
effective to provide a pH of less than about 6 (preferably, less than 
about 5, and more preferably, less than about 4). Alternatively, the 
present invention provides a kit for isolating RNA comprising instruction 
means for isolating substantially undegraded RNA from a biological sample 
and a Protein-DNA Precipitation Reagent comprising a sodium or potassium 
salt in an amount effective to precipitate DNA and protein, water, and an 
amount of a buffer effective to provide a pH of less than about 6. This 
reagent can be used in a method to isolate RNA from a biological sample 
containing substantially undegraded RNA released from cells or protein 
coats (i.e., a lysate) prepared using the Cell Lysis Reagent described 
above or a variety of known reagents for forming lysates. 
The kits of the present invention can also include an RNA Hydration Reagent 
comprising substantially RNase-free deionized water for hydrating RNA once 
it is isolated from a biological sample. For isolating RNA from mammalian 
whole blood, the kit can include an RBC Lysis Reagent comprising ammonium 
chloride, sodium bicarbonate, and EDTA. For isolating RNA from yeast and 
Gram-positive bacteria, the kit can include a Cell Suspension Reagent 
comprising tris[hydroxymethyl]aminomethane, EDTA, and sorbitol; and a 
Lytic Enzyme Reagent comprising a lytic enzyme, glycerol, 
tris[hydroxymethyl]aminomethane, and calcium chloride. 
The invention also provides a method for isolating RNA from a biological 
sample. This method involves contacting the biological sample with the 
Cell Lysis Reagent described above to lyse cells or protein coats to form 
a lysate containing substantially undegraded RNA. The substantially 
undegraded RNA is then separated from the lysate. This separation step 
preferably involves combining the lysate with the Protein-DNA 
Precipitation Reagent described above to precipitate DNA and protein. The 
substantially undegraded RNA is then separated from the precipitated DNA 
and protein to form substantially pure undegraded RNA. 
The present invention also provides a method for isolating RNA from 
mammalian blood comprising red and white blood cells. The method involves: 
contacting the blood with the RBC Lysis Reagent described above to lyse 
red blood cells and form a red cell lysate; separating the white blood 
cells from the red cell lysate; contacting the white blood cells (and any 
cell-associated viruses) with the Cell Lysis Reagent described above to 
lyse the cells and protein coats to form a white cell lysate containing 
substantially undegraded RNA; and separating the substantially undegraded 
RNA from the white cell lysate. This separating step preferably involves: 
combining the white cell lysate with the Protein-DNA Precipitation Reagent 
described above to precipitate DNA and protein; and separating the 
substantially undegraded RNA from the precipitated DNA and protein to form 
substantially pure undegraded RNA. 
A further embodiment of the invention is a method for isolating RNA from a 
biological sample, such as yeast or Gram-positive bacteria. The method 
involves: combining the biological sample with the Cell Suspension Reagent 
described above to form a cell suspension; adding the Lytic Enzyme Reagent 
described above to the cell suspension to form a mixture containing 
digested cells; separating the digested cells from the mixture; contacting 
the digested cells with the Cell Lysis Reagent to lyse the cells and 
protein coats to form a cell lysate containing substantially undegraded 
RNA; and separating the substantially undegraded RNA from the cell lysate. 
The separating step preferably includes combining the cell lysate with the 
Protein-DNA Precipitation Reagent to precipitate DNA and protein, and 
separating the substantially undegraded RNA from the precipitated DNA and 
protein to form substantially pure undegraded RNA. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides methods and kits that use aqueous reagents 
for isolating RNA from biological samples. Such biological samples include 
biological material, typically in an aqueous mixture, that contains RNA, 
including complex biological mixtures of procaryotic or eucaryotic cells. 
Typically, the biological material also includes DNA, proteins, and 
lipids. This includes, for example, biological fluids such as blood, 
saliva, and cerebrospinal fluid, solid animal tissues such as heart, 
liver, and brain, animal waste products such as feces and urine, plant 
tissues, yeasts, bacteria, viruses, mycoplasmas, fungi, protozoa, 
rickettsia, and other small microbial cells. 
Preferably, the methods and kits of the present invention provide 
substantially undegraded RNA. As used herein, "undegraded" RNA means 
nondigested or intact RNA, which can be readily determined by one of skill 
in the art using standard techniques. That is, the RNA is not damaged by 
enzymatic or chemical means during the isolation methods of the present 
invention. Preferably, the methods and kits of the present invention 
isolate a wide range of RNAs, such as ribosomal RNA, messenger RNA, 
transfer RNA, and viral RNA, all of which can be recovered over a wide 
molecular weight range. 
Using these methods and kits, RNA of substantially high yield can be 
obtained that is at least comparable to that obtained using conventional 
methods. Preferably, the isolated RNA is substantially pure, which can be 
determined by the absence of significant amounts of contaminating 
substances such as DNA and proteins, that could interfere with subsequent 
analyses, such as the sensitive assay reverse transcriptase-polymerase 
chain reaction (RT-PCR). Thus, the isolated RNA is suitable for use in 
subsequent analyses known to those of skill in the art. 
The process consists of cell or protein coat lysis and RNase inactivation 
by combining the biological material with a lysing reagent containing an 
anionic detergent at low pH to form a lysate. As used herein, "lysis" 
refers to the destruction of a cell by rupture of its membranes or 
envelope as well as the denaturation of a viral protein coat. This is 
followed by RNA purification using a high concentration, low pH salt 
reagent to selectively remove contaminating DNA and proteins. The final 
steps use common methods known to those of skill in the art. These steps 
are: (1) RNA concentration using standard precipitation methods; and (2) 
RNA hydration using a standard hydration solution, such as RNase free 
water. 
The reagents used in the methods and kits of the present invention contain 
generally less hazardous components than many conventional RNA isolation 
reagents. Although, lower alcohols (i.e., (C.sub.1 -C.sub.4)alkanols) may 
be used in concentrating RNA and/or removing residual salt, the aqueous 
reagents of the present invention are substantially free of organic 
solvents. As used herein, "substantially free" means less than about 1%, 
and typically less than 0.6%, volume/volume. Furthermore, they are at a 
lower pH than many conventional RNA isolation reagents. In addition, the 
reagents described in the present invention are much more stable than 
conventional RNA isolation reagents. Generally, the aqueous reagents of 
the present invention consist of aqueous formulations of common salts and 
detergents that are stable for at least about 18 months at room 
temperature (20-30.degree. C.). 
The first reagent, referred to herein as a "Cell Lysis Reagent," includes 
an anionic detergent dissolved in water. The reagent is buffered to a pH 
of less than about 6, preferably, less than about 5, and more preferably, 
less than about 4. The pH of all reagents described herein can be 
determined using a standard laboratory pH meter with no specific sample 
preparation. This reagent lyses cells and protein coats (e.g., as for 
viral RNA), and inactivates RNases rapidly enough that RNA is released 
from the cells or protein coats substantially undegraded, to form a 
lysate. Preferably, the pH of this reagent is at least about 2 and more 
preferably, at least about 3. 
Suitable anionic detergents are those that are soluble in water at a level 
of at least about 0.5% weight/volume, based on the total volume of the 
reagent, and are capable of lysing cells and/or solubilizing proteins and 
lipids at this concentration. Such anionic detergents include, but are not 
limited to, salts (e.g., sodium, potassium, and lithium salts) of dodecyl 
sulfate as well as N-lauroyl sarcosine. Preferably, the anionic detergent 
is a dodecyl sulfate salt. The anionic detergent is present in an amount 
effective to lyse cells and denature proteins causing the release of 
substantially undegraded RNA. Preferably, it is present in an amount of 
about 0.5-3%, more preferably, about 1-2.5%, and most preferably about, 
1.8-2.2% weight/volume, based on the total volume of the reagent. 
The pH of the Cell Lysis Reagent is maintained at less than about 6 using a 
buffer, such as a citrate buffer, although a citrate buffer is not a 
requirement as long as the buffer is capable of providing a pH of less 
than about 6 in aqueous media. For example, buffers such as acetate, 
glycine, phthalate, aconitate, and succinate can be used. Preferably, this 
pH is maintained using sodium citrate and citric acid in combination. 
Preferably, the molar ratio of sodium citrate to citric acid is about 
1:0.2 to about 1:13, and more preferably, about 1:2 for a pH of less than 
about 4. Preferably, a pH of less than about 6 is maintained using sodium 
citrate at about 10-100 mM, more preferably, 50-90 mM, and most 
preferably, 66-70 mM, concentration, based on the total volume of the 
reagent. Preferably, this pH is maintained using citric acid at 80-160 mM, 
more preferably, about 100-150 mM, and most preferably, about 130-134 mM, 
concentration, based on the total volume of the reagent. 
In addition to the anionic detergent and buffer, this first reagent 
includes a chelating agent. Suitable chelating agents are those capable of 
chelating divalent cations in aqueous media. Such chelating agents 
include, but are not limited to, ethylene diamine tetraacetate (EDTA) and 
cyclohexane diamine tetraacetate (CDTA). Preferably, the chelating agent 
is EDTA. A chelating agent is used in an amount effective to reduce DNase 
activity so that DNA is preferably released from the cells substantially 
undegraded to facilitate its subsequent removal. Preferably, the chelating 
agent is present in an amount of about 0.1-100 mM, more preferably about 
1-20 mM, and most preferably about 8-12 mM, based on the total volume of 
the reagent. 
The second reagent, referred to herein as a "Protein-DNA Precipitation 
Reagent," includes a sodium or potassium salt dissolved in water. The 
reagent is buffered to a pH of less than about 6, preferably, less than 
about 5, and more preferably, less than about 4. It is used for 
purification of the RNA. It includes a relatively high salt concentration, 
which causes contaminants such as DNA and protein to be selectively 
precipitated, thereby enabling them to be removed by centrifugation. 
Because of the relatively low pH, the RNA remains in solution. Preferably, 
the pH of the Protein-DNA Precipitation Reagent is at least about 2, and 
more preferably, at least about 2.5. 
Suitable salts for use in this second reagent are those that are soluble in 
water and are capable of causing precipitation of DNA and proteins from a 
lysate. Such salts include, but are not limited to, sodium salts such as 
sodium chloride and sodium acetate, potassium salts such as potassium 
chloride and potassium acetate. Preferably, the salt is sodium chloride. 
The salt is present in this Protein-DNA Precipitation Reagent in an amount 
effective to precipitate a sufficient amount of DNA and proteins out of a 
sample such that they do not interfere in the subsequent analysis of RNA. 
Preferably, the salt is present at a concentration of about 2-5.5 M, more 
preferably, at about 3-4.5 M, and most preferably, at about 3.8-4.2 M, 
based on the total volume of the reagent. 
The pH of the Protein-DNA Precipitation Reagent is maintained at less than 
about 6 using a buffer, such as a citrate buffer, although a citrate 
buffer is not a requirement as long as the buffer is capable of providing 
a pH of less than about 6 in aqueous media. For example, buffers such as 
acetate, glycine, phthalate, aconitate, and succinate can be used. 
Preferably, this pH is maintained using sodium citrate and citric acid in 
combination. Preferably, the molar ratio of sodium citrate to citric acid 
is about 1:0.2 to about 1:13, and more preferably, about 1:2 for a pH of 
less than about 4. Preferably, a pH of less than about 6 is maintained 
using sodium citrate at about 1-30 mM, more preferably, 10-25 mM, and most 
preferably, 15-20 mM, concentration. Preferably, this pH is maintained 
using citric acid at 10-60 mM, more preferably, about 20-50 mM, and most 
preferably, about 30-35 mM, concentration. 
Both the Cell Lysis Reagent and the Protein-DNA Precipitation Reagent 
include water and substantially no organic solvents or other similarly 
hazardous components. Preferably, the water is deionized. More preferably, 
the water is deionized to a level such that its resistance is greater than 
about 17 megohms. Most preferably, the water is deionized to this level of 
purity and further purified by filtering through a 0.2 .mu.M pore size 
filter. 
Another aspect of this invention involves the combination of the two 
reagents, Cell Lysis Reagent and Protein-DNA Precipitation Reagent, with 
one or more optional, ancillary reagents. These ancillary reagents include 
reagents known to one of skill in the art for nucleic acid purification. 
The methods and kits of the present invention, however, are not limited to 
the use of these specific ancillary reagents, as one of skill in the art 
may use other reagents and/or techniques to achieve the same purpose. 
Also, each of the Cell Lysis Reagent and the Protein-DNA Precipitation 
Reagent can be used with other reagents and/or techniques if desired. 
A first ancillary reagent is substantially RNase-free deionized water, 
which is used to hydrate the purified RNA at the final step in the RNA 
isolation process. This reagent is referred to herein as "RNA Hydration 
Reagent." It includes water deionized to a level such that its resistance 
is greater than about 17 megohms and further purified by filtering through 
a 0.2 .mu.M pore size filter. In addition, the deionized water is treated 
with diethylpyrocarbonate (DEPC), or similar such material, to inactivate 
RNases. Preferably, DEPC is initially present in the deionized water at a 
concentration of about 0.05-0.1%, and more preferably 0.06-0.07% 
(volume/volume), based on the total volume of the water. The DEPC is mixed 
with the deionized water and held at room temperature for about 3-24 
hours. Then the solution is heated under conditions effective to 
substantially completely decompose the DEPC to CO.sub.2 and ethanol. This 
typically occurs at a temperature of at least about 100.degree. C. and a 
pressure of at least about 20 psi (pounds per square inch) for at least 
about 40 minutes in a standard autoclave. Thus, when ready for use, the 
RNA Hydration Reagent is substantially free of organic components (i.e., 
less than about 1%, and typically less than about 0.6% volume/volume). 
The second ancillary reagent is a red blood cell lysing reagent used to 
lyse red blood cells and facilitate subsequent isolation of RNA from the 
white blood cells contained in mammalian whole blood. This reagent is 
referred to herein as the "RBC Lysis Reagent" and contains ammonium 
chloride, sodium bicarbonate, and EDTA. Preferably, the ammonium chloride 
is present in the RBC Lysis Reagent at a concentration of about 140-150 
mM, more preferably, at about 142-146 mM, based on the total volume of the 
reagent. Preferably, the sodium bicarbonate is present at a concentration 
of about 0.5-5 mM, and more preferably, at about 0.5-2 mM, based on the 
total volume of the reagent. Preferably, the EDTA is present at a 
concentration of about 0.5-10 mM, and more preferably, at about 0.75-1.25 
mM, based on the total volume of the reagent. RBC Lysis Reagent contains 
deionized water, preferably deionized to the level of purity described 
above, and further purified by filtration using a filter of about 0.2 
.mu.M pore size. 
When combined with mammalian whole blood, the RBC Lysis Reagent forms a red 
cell lysate containing substantially intact white blood cells. It can also 
contain viruses with substantially intact protein coats. The white blood 
cells (and any cell-associated viruses that may be present) are then 
separated from the red cell lysate. The Cell Lysis Reagent is then 
combined with the white blood cells to lyse the white cells and protein 
coats (of the cell-associated viruses) to form a white cell lysate. 
The third and fourth ancillary reagents are used together to isolate RNA 
from yeast and Gram-positive bacteria. The reagents are referred to herein 
as "Cell Suspension Reagent" and "Lytic Enzyme Reagent." They are used in 
the first steps of the RNA isolation procedure to digest cell walls as 
described in Example 6 below. The Cell Suspension Reagent is combined with 
a biological sample to form a cell suspension. The Lytic Enzyme Reagent is 
combined with the cell suspension to form a mixture containing digested 
cells. These digested cells are then separated from the mixture by 
centrifugation, for example, and then contacted with the Cell Lysis 
Reagent. 
The Cell Suspension Reagent preferably has a pH of about 7-8.5, and more 
preferably, about 7.5-8.0. It keeps cells intact while their cell walls 
are being digested by lytic enzyme. This reagent contains 
tris[hydroxymethyl]aminomethane (Tris), preferably, at a concentration of 
about 0.05-0.15 M, and more preferably, at about 0.08-0.12 M, based on the 
total volume of the reagent. The Cell Suspension Reagent also contains 
EDTA, preferably, at a concentration of about 0.05-0.15 M, and more 
preferably, at about 0.08-0.12 M, based on the total volume of the 
reagent. The preferred molar ratio of Tris to EDTA is about 1:1. This 
reagent also contains sorbitol, preferably at a concentration of about 
0.8-1.0 M, and more preferably, at a concentration of about 0.85-0.95 M, 
based on the total volume of the reagent. The Cell Suspension Reagent 
contains deionized water; preferably deionized to the level of purity 
described above, and further purified by filtration using a filter of 
about 0.2 .mu.M pore size. 
The Lytic Enzyme Reagent contains a lytic enzyme that digests 
beta-1,3-glucose polymers that are contained in yeast cell walls. A 
purified form of this enzyme is readily available from commercial sources 
such as Sigma Chemical Company, St. Louis, Mo. The activity of this enzyme 
is preferably at least about 200 units per mg, more preferably, at least 
about 1000 units per mg, and most preferably, at least about 5000 units 
per mg. This enzyme is dissolved in deionized water, preferably of the 
purity describe above containing preferably about 20-50% glycerol 
(volume/volume), more preferably about 25-40% glycerol and most preferably 
about 28-32% glycerol. In addition, the lytic enzyme reagent contains 
Tris, preferably, at a concentration of about 1-20 mM, more preferably, at 
about 5-15 mM, and most preferably, at about 8-12 mM, based on the total 
volume of the reagent. Finally, this reagent contains calcium chloride, 
preferably, at a concentration of about 0.5-5 mM, and more preferably, at 
about 0.75-1.25 mM, based on the total volume of the reagent. The pH of 
the lytic enzyme reagent is adjusted to a pH of about 7.5-8.2 using an 
acid such as hydrochloric acid, and purified by filtration through a 
filter of about 0.2 .mu.M pore size. 
As another aspect of this invention, a kit is provided that includes 
specific protocols, which in combination with the reagents described 
herein, may be used for isolating RNA according to the method of the 
invention. The kit includes the Cell Lysis Reagent and the Protein-DNA 
Precipitation Reagent. Depending on the application, the kit may also 
include one or more of the ancillary reagents described herein. The 
protocols may be scaled up or down depending upon the amount of biological 
material used provided the ratio of reagents remains consistent. Three 
particularly preferred RNA isolation kits provided are described below. 
A kit for isolating RNA from mammalian blood contains the RBC Lysis 
Reagent, the Cell Lysis Reagent, the Protein-DNA Precipitation Reagent, 
the RNA Hydration Reagent, and instruction means for isolating RNA from 
whole mammalian blood, preferably from 0.3 ml and 3 ml whole mammalian 
blood samples. A method to illustrate the use of this kit is described in 
Example 1. Using this kit, RNA is preferably isolated in a yield of at 
least about 0.5 .mu.g per 0.3 ml whole blood, and typically in a range of 
about 0.5-2.5 .mu.g per 0.3 ml whole blood; however, the yield depends on 
the white cell count which varies considerably from individual to 
individual. 
A kit for isolating RNA from plant and animal solid tissues, cultured plant 
and animal cells, body fluids such as cerebrospinal fluid, plasma, saliva, 
semen, serum, synovial fluid, urine, or Gram-negative bacteria contains 
the Cell Lysis Reagent, the Protein-DNA Precipitation Reagent, the RNA 
Hydration Reagent, and instruction means for isolating RNA from, for 
example, 5-10 mg and 50-100 mg plant and animal solid tissue samples, 1-2 
and 10-20 million cultured plant and animal cells, 100 .mu.l body fluids, 
0.5 ml (0.5 billion cell) or 5 ml (5 billion cell) Gram-negative bacteria. 
Methods to illustrate the use of this kit are given in Examples 2-5. Using 
this kit, RNA is preferably isolated in a yield of at least about 0.5 
.mu.g per 1 mg plant or animal solid tissue, and typically in a range of 
about 0.5-6 .mu.g per 1 mg plant or animal solid tissue; at least about 5 
.mu.g per million cultured plant and animal cells, and typically in a 
range of about 5-10 .mu.g per million cultured plant and animal cells; and 
at least about 7 .mu.g per 0.5 ml overnight culture of Gram-negative 
bacteria, and typically in a range of about 7-15 .mu.g per 0.5 ml 
overnight culture of Gram-negative bacteria. 
A kit for isolating RNA from yeast and Gram-positive bacteria contains the 
Cell Suspension Reagent, the Lytic Enzyme Reagent, the Cell Lysis Reagent, 
the Protein-DNA Precipitation Reagent, the RNA Hydration Reagent, and 
instruction means for isolating RNA from yeast or bacterial cells, 
preferably from 1 ml or 1-2.times.10.sup.8 and 10 ml or 
10-20.times.10.sup.8 yeast cells, or 0.5 ml or 0.5 billion and 5 ml or 5 
billion Gram-positive bacteria cells. A method to illustrate the use of 
this kit is described in Example 6. Using this kit, RNA is preferably 
isolated in a yield of at least about 150 .mu.g per 1 ml yeast overnight 
culture, and typically about 150-275 .mu.g per 1 ml yeast overnight 
culture; and at least about 1 .mu.g per 0.5 ml Gram-positive bacteria 
overnight culture, and typically about 1-4 .mu.g per 0.5 ml Gram-positive 
bacteria overnight culture. 
The methods of the present invention involve combining a biological sample 
with the Cell Lysis Reagent to form a lysate containing substantially 
undegraded RNA. The Protein-DNA Precipitation Reagent is typically then 
added directly to this lysate to selectively precipitate contaminants such 
as proteins and DNA. The supernatant is then collected and substantially 
undegraded RNA is precipitated from the supernatant by the addition of a 
lower alcohol. The precipitated RNA is then recovered by centrifugation 
and decanting. It is then generally washed with a lower alcohol and dried. 
The RNA is then typically rehydrated with the RNA Hydration Reagent. As 
discussed above, the initial biological sample may be pretreated with one 
or more of the other ancillary reagents. 
The invention will be further described by reference to the following 
detailed examples. These examples are offered to further illustrate the 
various specific and illustrative embodiments and techniques. It should be 
understood, however, that many variations and modifications may be made 
while remaining within the scope of the present invention. All of the raw 
materials mentioned below are readily available from commercial sources 
such as Sigma Chemical Company, St. Louis, Mo. All percentages are in 
weight per volume, based on the total volume of the reagent, unless 
specified otherwise. RNA yields were measured using standard ultraviolet 
spectrophotometry techniques.

EXAMPLES 
Example 1 
RNA Isolation from Human Whole Blood 
Sample Preparation: White cells from three samples of human whole blood 
were used as a source of RNA. White cells were prepared by adding 0.3 ml 
whole blood to the RBC Lysis Reagent, which preferentially lyses red cells 
during a 10 minute incubation at room temperature. This reagent contained 
144 mM ammonium chloride, 1 mM sodium bicarbonate, and 1 mM EDTA. White 
cells were collected by centrifuging at 15,000.times. g for 20 seconds and 
discarding all but 10-20 .mu.l of the supernatant fraction. The white cell 
pellet was vortexed for several seconds to suspend the cells in the 
residual supernatant fluid. 
Sample Processing: To the cells, 0.3 ml Cell Lysis Reagent was added; this 
aqueous solution contained 2% sodium dodecyl sulfate, 68 mM sodium 
citrate, 132 mM citric acid, 10 mM EDTA. The cells were lysed by pipeting 
the lysis solution up and down not more than three times. Then 100 .mu.l 
Protein-DNA Precipitation Reagent was added; this aqueous solution 
contained 17 mM sodium citrate, 33 mM citric acid, and 4 M sodium 
chloride. The Protein-DNA Precipitation Reagent was mixed into the lysate 
by inverting the sample 10 times. This mixture was placed on ice for 5 
minutes and then centrifuged for 3 minutes at room temperature to sediment 
contaminating DNA and proteins. The supernatant fraction containing the 
purified RNA was added to a clean tube containing 300 .mu.l isopropanol to 
precipitate the RNA. RNA was sedimented by centrifuging the sample at 
15,000.times. g for 3 minutes and the supernatant discarded. The RNA 
pellet was washed by adding 0.3 ml 70% ethanol, centrifuging at 
15,000.times. g for 1 minute and then pouring off the supernatant 
fraction. The tube was inverted on clean absorbent paper and the RNA 
pellet was air dried for 10 minutes. Finally, the RNA was rehydrated by 
incubating on ice for 30 minutes in 50 .mu.l RNA Hydration Reagent 
(RNase-free deionized water) and stored at -80.degree. C. 
Sample Yields: The yields of RNA from white cells were 1.5 .mu.g, 1.2 
.mu.g, and 0.9 .mu.g for the three 0.3 ml whole blood samples. 
Example 2 
RNA Isolation from Cultured Mammalian Cells 
Sample Preparation: Cultured mammalian cells (D17 dog cells) were used as 
the source of RNA for this example. One half million cells in culture 
medium were sedimented by centrifuging at 15,000.times. g for 5 seconds 
and all but 10-20 .mu.l of the supernatant were discarded. The cell pellet 
was vortexed for several seconds to suspend the cells in the residual 
supernatant fluid. 
Sample Processing: Samples were processed following the method described in 
Example 1. 
Sample Results: The yields of RNA from cultured mammalian cells were 6.2 
.mu.g, 4.2 .mu.g, and 4.3 .mu.g for the three 0.5 million cell samples. 
Example 3 
RNA Isolation from Animal Tissue (Drosophila melanogaster) 
Sample Preparation: Three samples each containing five adult male and five 
adult female flies were used as a source of RNA. The flies were 
immobilized by cooling to 4.degree. C., transferred to tubes and then 
weighed. 
Sample Processing: Each tube was kept on ice until adding 0.3 ml Cell Lysis 
Reagent. The flies were homogenized in the Cell Lysis Reagent using a 
pestle. The remaining steps follow those described in Example 1. 
Sample Results: The yields of RNA from each of the three 10 fly 
preparations were: 17.0 .mu.g from 9 mg tissue, 14.7 .mu.g from 6 mg 
tissue and 11.8 .mu.g from 7 mg tissue. The average relative yield was 2.0 
.mu.g RNA per mg tissue. 
Example 4 
RNA Isolation from Plant Tissue (alfalfa) 
Sample Preparation: Three samples each containing five alfalfa cotyledons 
(first leaves) were used as a source of RNA. The leaves were cooled to. 
4.degree. C., transferred to tubes and then weighed. 
Sample Processing: Each tube was kept on ice until adding 0.3 ml Cell Lysis 
Reagent. The leaves were homogenized in the Cell Lysis Reagent using a 
pestle. The remaining steps follow those described in Example 1. 
Sample Results: The yields of RNA from each of the three 11 mg alfalfa 
samples were: 13.3 .mu.g, 12.7 .mu.g, and 9.9 .mu.g. The average relative 
yield was 1.1 .mu.g RNA per mg tissue. 
Example 5 
RNA Isolation from Escherichia coli bacteria 
Sample Preparation: Cultured bacterial cells (standard DH5.alpha..TM. cells 
Life Technologies, Inc., Gaithersburg, Md.) were used as the source of RNA 
for this example. Approximately 0.5 billion cells in 0.5 ml culture medium 
were sedimented by centrifuging at 15,000.times. g for 5 seconds and all 
but 10-20 .mu.l of the supernatant fraction were discarded. The cell 
pellet was vortexed for several seconds to suspend the cells in the 
residual supernatant fluid. 
Sample Processing: Samples were processed following the method described in 
Example 1 except that the cell lysate was incubated at 65.degree. C. for 5 
minutes to complete cell lysis. 
Sample Results: The yield of RNA from each of the three 0.5 ml bacterial 
cell samples was 13.0 .mu.g, 14.6 .mu.g, and 8.0 .mu.g. The average 
relative yield was 23.7 .mu.g RNA per 1 ml overnight culture. 
Example 6 
RNA Isolation From Yeast (Saccharomyces cerevisiae) 
Sample Preparation: Cultured yeast cells were used as the source of RNA for 
this example. Three samples containing approximately 100 million cells in 
culture medium were sedimented by centrifuging at 15,000.times. g for 5 
seconds and all but 10-20 .mu.l of the supernatant fraction were 
discarded. The cell pellet was suspended in the Cell Suspension Reagent 
containing 0.1 M Tris, 0.1 M EDTA, and 0.9 M sorbitol. Then 1.5 .mu.l 
lytic enzyme reagent, containing 4000 units per .mu.l lytic enzyme 
dissolved in 30% glycerol (volume/volume), 10 mM Tris and 1 mM calcium 
chloride, were added to digest the yeast cell walls. After incubation at 
37.degree. C. for 30 minutes, the cells were centrifuged at 15,000.times. 
g for 1 minute and the supernatant fraction was removed. 
Sample Processing: Samples were processed following the method described in 
Example 1. 
Sample Results: The yields of RNA from the three 1 ml overnight cultures of 
yeast cells were 168.0 .mu.g, 167.6 .mu.g, and 165.6 .mu.g. 
Example 7 
Analysis of Total RNA Using Agarose Gel Electrophoresis 
To assess the quality of isolated RNA, 500 nanogram samples were 
electrophoresed through a 1.5% agarose gel at 80 volts for 40 minutes. 
Total RNA was isolated successfully from cultured hepatocarcinoma cells, 
Japanese quail liver, fruit fly (Drosophila melanogaster), alfalfa leaf, 
yeast (Saccharomyces cerevisiae), and Gram-negative bacteria (Escherichia 
coli). The presence of intact ribosomal RNA bands in all of the samples 
indicated that the isolated RNA contained substantially undegraded, high 
quality total RNA ranging from high to low molecular weight. 
Example 8 
Analysis of RNA Using RT-PCR 
To further assess the quality of RNA, a reverse transcription (RT) 
polymerase chain reaction (PCR) assay was performed using methods 
essentially as described in Kohler et al., Leukemia, 4, 541-547 (1990). 
Total RNA isolated from two human whole blood samples using the method of 
this invention, described in Example 1, was used for this assay. RNA 
samples of 100 ng were treated with reverse transcriptase to generate a 
DNA copy. Then oligonucleotide primers specific for the c-abl 
proto-oncogene were used to amplify this DNA during 35 cycles, where a 
cycle was defined as 94.degree. C. for 1 minute, 55.degree. C. for 1 
minute and 72.degree. C. for 2 minutes. The amplified DNA was 
electrophoresed through a 3% agarose gel at 80 volts for 1 hour. A band of 
290 base pairs indicated an amplification product derived from RNA while a 
band of 920 base pairs was expected if excess contaminating DNA were 
present in the RNA samples. This assay showed the presence of 
substantially pure RNA. An RT-PCR amplification product of 920 base pairs 
in size was not detected in either sample, indicating the absence of 
substantial contaminating DNA. However, an RT-PCR amplification product of 
290 base pairs in size was observed in both samples, indicating the 
presence of substantially undegraded RNA. In addition, the presence of an 
amplification product for each sample showed that whole blood contaminants 
such as protein and heme, which inhibit this reaction, were substantially 
removed by the purification method. 
The complete disclosure of all patents, patent documents, and publications 
cited herein are incorporated by reference. The foregoing detailed 
description and examples have been given for clarity of understanding 
only. No unnecessary limitations are to be understood therefrom. The 
invention is not limited to the exact details shown and described, for 
variations obvious to one skilled in the art will be included within the 
invention defined by the claims.