Nucleic acid purification

Abstract

Methods and composition for nucleic acid isolation are provided. In one embodiment, a method is provided for nucleic acid purification from biological samples, such as whole blood samples, extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for of small RNAs (e.g., siRNAs or miRNAs) purification and are amenable to automation.

Claims

1. A method for binding a ribonucleic acid (RNA) to a silica substrate for purification comprising: (a) contacting a sample comprising whole blood or blood cells with a denaturing solvent comprising a guanidinium thiocyanate and at least 20% to 60% phenol, the denaturing solvent having a pH of 3.5-6.0; (b) adding a binding agent to the sample, wherein the binding agent comprises a chaotropic salt, a lower alcohol or a mixture thereof; (c) contacting the sample with a silica substrate, in the presence of at least 10% to 50% phenol, thereby binding the ribonucleic acid in the sample to the silica substrate; (d) washing the silica substrate and bound RNA with a wash solution; and (e) eluting the RNA from the silica substrate with an elution buffer, thereby providing purified RNA; wherein at least 90% of the RNA in the sample is unprecipitated prior to binding of the ribonucleic to the silica substrate, and wherein after the addition of the binding agent, the sample does not comprise separate aqueous and organic phases prior to binding the ribonucleic acid to the silica substrate.

2. The method of claim 1, wherein the denaturing solvent comprises at least one additional agent selected from the group consisting of a chaotropic salt, an antioxidant, a chelating agent, and a buffer.

3. The method of claim 1, wherein the method is automated.

4. The method of claim 1, wherein the binding agent is added to the sample before the denaturing solvent.

5. The method of claim 1, wherein the silica substrate is added to the sample before the binding agent.

6. The method of claim 1, wherein steps (a) and (b) are performed contemporaneously.

7. The method of claim 6, wherein steps (a), (b) and (c) are performed contemporaneously.

8. The method of claim 1, wherein the binding agent comprises a lower alcohol selected from the group consisting of methanol, ethanol, isopropanol, butanol, and a combination thereof.

9. The method of claim 1, wherein the total alcohol content of the sample is at least 40% after the binding agent is added.

10. The method of claim 1, wherein the binding agent is an aqueous solution comprising a chaotropic salt or mixture of chaotropic salts.

11. The method of claim 10, wherein the chaotropic salt is guanidinium thiocyanate, guanidinium chloride, sodium iodide, sodium perchlorate, urea or thiourea.

12. The method of claim 10, wherein the binding agent comprises pH buffer.

13. The method of claim 1, wherein steps (b) and (c) are performed contemporaneously.

14. The method of claim 1, wherein the ribonucleic acid is miRNA or siRNA.

15. The method of claim 1, wherein the silica substrate is in the form of beads, fibers or a porous matrix.

16. The method of claim 1, wherein the silica substrate is a (para)magnetic bead.

17. The method of claim 1, wherein the wash solution comprises a salt, a chaotropic salt, a detergent or an alcohol.

18. The method of claim 1, wherein in step (a) the guanidinium thiocyanate is present at a concentration of 0.5 M to 2 M.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1: A non-limiting exemplary protocol for isolation of nucleic acid according to the invention.

(3) FIG. 2: Visualization of RNA isolated in the studies of Example 1 following electrophoresis on a 1% agarose gel. Treatment conditions for the samples loaded in lanes 1-11 (from left to right) are as follows (1) 1 KB ladder; (2) RNAzol+H.sub.2O+isopropanol; (3) RNAzol+isopropanol; (4) RNAzol+H.sub.2O+ethanol; (5) RNAzol+ethanol; (6) RNAzol-control; (7) TRIreagent+isopropanol; (8) TRIreagent+chloroform+ethanol; (9) TRIreagent+ethanol; (10) TRIreagent-control; (11) Quick-RNA-control, see also Table 1.

(4) FIG. 3: Visualization of nucleic acid isolated in the studies of Example 2 following electrophoresis on a 1% agarose gel. Treatment conditions for the samples loaded in lanes 1-11 (from left to right) are as follows (1) 1 KB ladder; (2) buffer D; (3) buffer A; (4) buffer B; (5) column flow-through from lane 4 sample; (6) buffer C; (7) column flow-through from lane 6 sample; (8) ethanol; (9) isopropanol; (10) 1 KB ladder; (11) buffer C+ethanol, see also Table 2.

(5) FIG. 4A-B: Reproducible automated sample processing. Isolation of RNA from phenol-containing sample lysates, without phase separation, allows for direct binding of nucleic acids to a column matrix or bead and for automated processing. A, Direct-zol MagBead RNA Isolation. Graph shows a comparison between manual and automated (Freedom EVO, Tecan) sample processing by nucleic acid binding directly from a phenol-containing denaturing reagent using the Direct-zol-96 MagBead RNA across a 96-well plate. In each case, RNA was purified from human epithelial cells (510.sup.5/well). B, RNA was purified from human epithelial cells using the Direct-zol-96 MagBead RNA on a Freedom EVO automated system. RNA quality was assessed using a Bioanalyzer. RNA integrity number (RIN) demonstrates the high quality of the isolated RNA.

(6) FIG. 5A-B: Isolation of small RNAs. Direct binding of RNA and small RNAs from phenol-containing lysates without phase separation, increases recovery of small RNAs compared to the conventional method (i.e., with phase separation/precipitation). A, Graph shows direct RNA isolation from phenolic extracts using a column capture, without phase separation (gray bars) versus a conventional phase separation method (lights bars). Methods of the embodiments show 4 higher recovery of RNAs<40 nt as compared to a conventional phase separation method. B, Efficient small RNA isolation using a bead capture. Small RNA was recovered with the Direct-zol-96 MagBead RNA system and gel-separated and imaged using a Bioanalyzer. Sample 1, indicates small RNA recovery from total RNA. Sample 2, indicates small RNA alone. Both samples were spiked with small-RNA ladder prior to Direct-zol MagBead purification.

(7) FIG. 6: Isolation of nucleic acid from blood samples. Both DNA and RNA can be efficiently isolated from phenol-containing whole-blood lysates directly, without phase separation. Gel shows nucleic acids efficiently isolated from whole blood in TRI Reagent without a phase separation. 100 l whole blood (pig) was lysed in 300 l TRI Reagent, cleared by centrifugation and nucleic acids bound to silica beads (Direct-zol MagBead system).

(8) FIG. 7: Isolation of RNA from human cells is shown. High quality RNA is isolated from different binding buffers comprising different alcohols or mixtures of alcohols or Dioxane without phase separation.

(9) FIG. 8: Isolation of RNA from human cells is shown. High quality RNA is isolated from different binding buffers comprising different chaotropic salts.

DETAILED DESCRIPTION

(10) The instant invention provides an efficient method for nucleic acid isolation from samples. In particular, methods of the invention allow the binding of nucleic acids directly from an organic sample suspension to a silica substrate. For example, by adding an aqueous binding buffer to an organic suspension DNA can be efficiently bound to a silica matrix and purified away from contaminating protein and cell debris. Likewise, an alcohol based binding buffer can be used to directly bind RNA (and DNA) to silica from an organic suspension. Thus, the methods of the instant invention can be used to purify RNA (including small RNA molecules such as siRNA and miRNA), DNA, DNA and RNA or to preferentially purify RNA and/or DNA

(11) The direct binding of nucleic acids from an organic suspension offers a number of advantages relative to other protocols for purification. First, the organic and aqueous phases of a suspension do not need to be separated, which is time consuming and typically involves centrifugation of the suspension and laborious removal of the aqueous (or organic) phase. Moreover, because any residual phenol contamination can inhibit the effectiveness of downstream sample treatments (e.g., nucleic acid sequencing), one or more additional step involving extracting the sample with a further organic compound is often required to reduce phenol contamination. Second, there is no need to precipitate nucleic acid out of solution. Again, this process is labor intensive and, in the case of samples containing small amounts of nucleic acid, can result in almost complete loss of the sample's nucleic acid. The direct binding of nucleic acids also has the advantage of reducing the opportunity for nuclease attack of the nucleic acids from the sample. In particular, because the methods of the invention do not involve additional precipitation and centrifugation steps in aqueous buffers the period over which the nucleic acids could be exposed to any active contaminating nuclease is reduced. Likewise, because the sample does not need to be transferred to multiple containers the chance of importing exogenous nuclease is reduced. The foregoing advantages of the new purification methods make them ideal for modern high throughput protocols which require reduced labor input.

I. GENERAL PROTOCOL

(12) An illustrative and non-limiting protocol for nucleic acid purification according to the invention is exemplified below.

(13) A. Sample Extraction

(14) Samples may be processed for example, by freeze-thaw, proteinase treatment or mechanical homogenization prior to organic extraction. Organic extraction may be accomplished with a protein denaturing reagent such as a phenol composition or acidic phenol (carbolic acid) composition with guanidinium thiocyanate, to form a suspension. Protein denaturing reagents may additionally comprise components such as a phase separating agent (e.g., chloroform), an antioxidant (e.g., 2-mercaptoethanol or lipoic acid) or a chelating agent. An example composition can comprise phenol:guanidinium thiocyanate:other component(s) (at a ratio of 5:3:2). Once the denaturing solvent is added, the sample may be further homogenized, for example, by vigorous shaking or blending to solubilize all possible cell components. An organic and aqueous phase may form in a sample which comprises phenol, but such phase separation is not required for purification.

(15) B. Addition of Binding Agent

(16) A binding agent is added to the sample. In particular the binding agent comprises a chaotropic salt, an alcohol, Dioxane or a mixture thereof that facilitates nucleic acid binding to a silica substrate. In the case of DNA, the binding agent is a substantially aqueous agent with chaotropic salt(s). A binding agent may also comprise additional buffer agents, salts, detergents and/or alcohol (e.g., lower alcohols such as ethanol or isopropanol). For example, the binding agent may comprise 4-5 M guanidinium thiocyanate (GTC), 5-20% isopropanol, 2-10% glycerol and detergents. In another example, the binding agent comprises 4-5 M GTC, 0.1-1.0 M sodium acetate and, optionally, detergents. One to six volumes of the binding agent are typically added to one volume of a phenol containing sample (e.g., 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6 volumes of binding agent:sample+phenol). In the case of RNA (or RNA/DNA), the binding agent is a substantially alcohol agent, such as ethanol or isopropanol. After addition of a substantially alcohol agent the total concentration of alcohol in the sample is typically raised to greater than about 20%. For embodiments where binding of both DNA and RNA is desired a binding buffer may comprise both a chaotropic salt, such as GTC, and an alcohol.

(17) C. Nucleic Acid Binding to Silica

(18) The sample comprising the binding agent is then contacted to a silica substrate. If an aqueous phase has formed in the sample then the aqueous phase alone may be contacted with the silica substrate. However, separation of organic and aqueous phases is not required for binding. The term silica as used herein refers materials comprising a build-up of silicon and oxygen. Such materials include, without limitation, silica, silicon dioxide, silica gel, fumed silica gel, diatomaceous earth, celite, talc, quartz, glass, glass particles including all different shapes of these materials. Particles, for example, may comprise particles of crystalline silica, soda-lime glasses, borosilicate glasses, and fibrous, non-woven glass. In certain aspects, a silica bead may be a magnetic silica bead (see, e.g., PCT Patent Publn. WO 98/31840, incorporated herein by reference).

(19) Following binding of the nucleic acid the remaining sample suspension is removed by, for example, pipette, vacuum or centrifugation.

(20) D. Optional Addition of a Second Binding Agent

(21) In certain cases, DNA can be bound to silica, while the majority of RNA remains unbound in the sample. In this case, the remaining sample can be collected and a second binding agent added. The binding agent can be a substantially alcohol agent or Dioxane agent for binding RNA such that, after addition, the total concentration of alcohol in the sample is raised to greater than about 20%, such as to a concentration of about 50% or higher.

(22) E. Optional Second Binding to Silica

(23) The sample and binding agent are contacted with a second silica substrate (as above) to bind additional remaining nucleic acid, such as RNA, from the sample. The remaining sample suspension is then removed as in step 3. The remaining sample may be held for further analysis, such as protein or lipid analysis, or discarded.

(24) F. Wash of Silica Substrate with Bound Nucleic Acid

(25) The silica substrate (and/or second silica substrate) comprising bound nucleic acid is washed one or more times with a washing agent. Washing agents may comprise, for example, solutions comprising alcohol, salts, buffering agents and/or detergents, that do not elute substantial amounts of nucleic acid from the silica. For example, a wash buffer may comprise about or greater than about 40%, 50%, 60%, 70%, 80% or 90% alcohol. In certain aspects the alcohol is a volatile alcohol, such as ethanol. Example washing agents include, but are not limited to, a solution of 20%-50% ethanol and 20%-50% isopropanol; a solution of about 0.1-4.0 M guanidinium hydrochloride, detergents and up to about 80% ethanol; or a solution of about 80% ethanol.

(26) G. Elute Nucleic Acid from the Silica Substrate

(27) The silica substrate (and/or second silica substrate) comprising bound nucleic acids is contacted with an elution buffer to remove the bound nucleic acid into solution. Elution buffers typically comprise a pH buffer agent, limited levels of salts and/or chelating agents. Buffer may additionally comprise nuclease inhibitors.

(28) Following elution nucleic acids may either be further purified or used directly in down-stream analysis such as hybridization or sequencing.

II. REAGENTS AND KITS

(29) Kits may comprise suitably aliquoted reagents of the present invention, such as an acid-phenol denaturing solvent, one or more binding agent and a silica substrate. Additional components that may be included in a kit according to the invention include, but are not limited to, one or more wash buffer including a magnetic bead (i.e., magnetic silica beads such as MagBinding Beads) Pre-wash buffer, an elution buffer, a proteinase composition, DNase and/or RNase inhibitors, DNase or RNase enzymes, oligonucleotide primers, reference samples (e.g., samples comprising known amounts of DNA or RNA), distilled water, DEPC-treated water, probes, sample vials, polymerase, magnetic binding beads (e.g., magnetic silica beads such as MagBinding Beads), 96-well silica plates, 96-well collection plates, cover foils for 96 well plates and instructions for nucleic acid purification. In certain further aspects, additional reagents for DNA and/or RNA clean-up may be included.

(30) The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing reagent containers in close confinement for commercial sale. Such containers may include cardboard containers or injection or blow-molded plastic containers into which the desired vials are retained.

(31) When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

III. EXAMPLES

(32) The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Example 1: RNA Isolation from Human Cells

(33) Various techniques were used for purification of RNA from human cells that were frozen at 80 C. RNA was isolated from 100,000 frozen human cells resuspended in 50 l of water. Samples in lanes 2-6 were lysed in 125 l of RNAzol and further were added one or more of the following, water, ethanol, isopropanol (indicated as Treatment in the Table 1). Samples in lanes 7-10 were lysed in 200 l TRIreagent and further were added one or more of the following, chloroform, ethanol, isopropanol (indicated as Treatment in the Table 1.). Samples in lanes 2-5 and 7-9 were processed by column purification using a silica matrix. Each suspension mixture including all components as indicated in Treatment (Table 1) was loaded onto the Zymo-Spin IC column (Zymo Research, Irvine Calif.), centrifuged at 12,000g, and flow-through discarded. The column was washed once with 700 l RNA Wash Buffer then centrifuged in an empty collection tube to remove residual ethanol. RNA was eluted from the column with 15 l water. Samples 6 and 10 are controls and were processed according to manufacturer's instructions, i.e., to separate aqueous and organic phase, samples were centrifuged. The aqueous phase was then precipitated with isopropanol, RNA pellet washed twice with 70% ethanol, then resuspended in 15 l water. Sample in lane 11 was lysed in 150 l of ZR RNA Buffer and processed according to the protocol for Quick-RNA MicroPrep (Catalog number R1050, Zymo Research, Irvine Calif.).

(34) Any RNA eluted after the indicated treatments was resolved by electrophoresis on a 1% agarose gel. Results from this study are shown in FIG. 2. Specifically, the results demonstrate that by using a binding agent comprising alcohol (such as ethanol or isopropanol) RNA could be efficiently bound to (and later eluted from) a silica matrix directly from the RNAzol or TRIreagent suspension (i.e., without the need for nucleic acid precipitation).

(35) TABLE-US-00001 TABLE 1 Sample treatments for RNA shown in FIG. 2 Lane Label Treatment 1 1kb 1kb DNA marker 2 RNAzol + H.sub.2O + +water (70 l) + isopropanol (245 l) isopropanol 3 RNAzol + +isopropanol (175 l) isopropanol 4 RNAzol + H.sub.2O + +water (70 l) + 95% ethanol (245 l) ethanol 5 RNAzol + ethanol +95% ethanol (175 l) 6 RNAzol/control +water (70 l) 7 TRIreagent + +isopropanol (250 l) isopropanol 8 TRIreagent + +chloroform (50 l) + 95% ethanol chloroform + ethanol (300 l) 9 TRIreagent + ethanol +95% ethanol (250 l) 10 TRIreagent/control +chloroform (50 l) + isopropanol (100 l) 11 Quick-RNA- control

Example 2: DNA and RNA Isolation from Human Cells

(36) Various techniques were used for purification of nucleic acid molecules from human cells that were frozen at 80 C. Frozen cell samples from 50,000 human cells were resuspended in 25 l of water. All samples (lanes 2-9 and 11) were lysed in 100 l of TriReagent. Following the lysis, a binding agent was added to the sample as indicated under Treatment (Table 2). The mixtures including all components as indicated in Treatment (Table 2) were loaded onto the Zymo-Spin IC column (Zymo Research, Irvine Calif.), centrifuged at 12,000g, and flow-through discarded (sample/lane 2-3, 5, 7-9, 11) or saved for further processing (sample/lane 4 and 6). 95% ethanol was added to the flow-through from samples/lanes 4 and 6 and the mixture was loaded onto the a new Zymo-Spin IC column, centrifuged at 12,000g, and flow-through discarded. After binding, all columns were washed once with 700 l RNA Wash Buffer then centrifuged in an empty collection tube to remove residual ethanol. RNA, DNA or DNA and RNA was eluted from the column with 15 l water. Buffer used in the experiments are as follows: Buffer A (A; 4-5 M GTC, 5-20% isopropanol, 2-10% glycerol and detergents); Buffer B (B; 4-5 M GTC and 0.1 to 1.0 M sodium acetate); Buffer C (C; 4-5 M GTC, 0.1 to 1.0 M sodium acetate and detergents); and Buffer D (D; 1-5M GTC, 1-7M NaI, 20-60% ethanol and detergent).

(37) Any nucleic acid that was eluted from the column was resolved by electrophoresis on a 1% agarose gel. Results from this study are shown in FIG. 3 and demonstrate that both DNA and RNA could be directly bound to a silica matrix directly from the organic denaturing agent (such as TRIreagent) suspension, without the need for nucleic acid precipitation. Furthermore, the results demonstrate that by using binding agents having different compositions DNA and RNA can be sequentially purified from sample. See, for example lanes 4 and 5 or 6 and 7 which show DNA and RNA isolated from the same sample, respectively.

(38) TABLE-US-00002 TABLE 2 Sample treatments for results shown in FIG. 3 Lane Label Treatment 1 1 kb 1 kb DNA marker 2 D +Buffer D (375 l) 3 A +Buffer A (375 l) 4 B +Buffer B (375 l) 5 B/FT Sample/lane 4 column flow-through + 95% ethanol (500 l) 6 C +Buffer C (375 l) 7 C/FT Sample/lane 6 column flow-through + 95% ethanol (500 l) 8 Ethanol +95% ethanol (125 l) 9 Isopropanol +Isopropanol (125 l) 10 1 kb 1 kb DNA marker 11 B + Ethanol +Buffer B (250 l) + 95% ethanol (375 l)

Example 3: Automated Nucleic Acid Isolation using Magnetic Beads

(39) Isolation of RNA (DNA) from phenol-containing sample lysates, without phase separation, allows for direct binding of nucleic acids to a column matrix or beads, such as magnetic beads, and is ideal for automated sample processing. As an example of such automated processing samples were processed as detailed below the resulting RNA quantified and assess for quality.

(40) 1. 510.sup.5 human epithelial cells were lysed in TRI Reagent and cleared by centrifugation.

(41) 2. Direct-zol Binding Buffer (see above) were added to the sample lysate (1:1 ratio) and mixed.

(42) 3. Magnetic silica beads (MagBinding Beads) were then added to the mixture to bind nucleic acids.

(43) 4. Beads are washed by 95-100% ethanol, Direct-zol MagBinding Bead PreWash, and 95-100% ethanol again, several times.

(44) 5. Optionally, during the wash steps, DNase I treatment can be performed with provided DNase I/10 Reaction Buffer.

(45) 6. Beads are dried and DNase/RNase-Free Water is added to elute RNA or DNA/RNA.

(46) As shown in FIG. 4 methods of the embodiments proved highly effective in the context of automated sample processing. FIG. 4A demonstrates that reproducibly high concentration, high yield RNA can be obtained by automated sample processing according to the embodiments. Likewise, as shown in FIG. 4B the RNA obtained is of reproducibly high quality and shows little or no significant degradation.

Example 4: Small RNA Molecule Recovery

(47) Experiments were undertaken to assess the efficiency of isolating small RNAs using methods of the embodiments. An example protocol for small RNA isolation is shown below:

(48) 1. RNA from human epithelial cells or mouse liver tissue homogenized in TRI Reagent was isolated by Direct-zol method and compared to the conventional phase separation. RNA was analyzed and quantified by a Small RNA Chip (Bionalyzer; FIG. 5).

(49) 2. A mixture of small RNA oligonucleotides (ZR small-RNA ladder) was recovered directly or spiked into a total RNA and recovered by the Direct-zol MagBinding Bead protocol. RNA was analyzed and quantified by a Small RNA Chip (Bioanalyzer; FIG. 5).

(50) Results of the studies shown in FIG. 5A demonstrate that direct RNA isolation from phenolic extracts, without phase separation, shows higher recovery of RNAs<40 nt (4 fold higher) as compared to a conventional phase separation method. Again recovered RNA was shown to be of high quality (FIG. 5B).

Example 5: Nucleic Acid Extraction from Whole Blood

(51) Both DNA and RNA can be isolated from phenol-containing whole-blood lysates directly, without phase separation. For these studies 100 ul of whole-blood (pig or human) was lysed in 300 ul TRI Reagent BD (TRI Reagent BD is for blood, plasma, serum) and cleared by centrifugation. The lysate/supernatant is aspirated and dispensed into a new tube. The lysate/supernatant can be processed either by spin-column/spin-plate (Direct-zol RNA MiniPrep, Direct-zol-96 RNA) or magnetic silica bead format (Direct-zol MagBinding Bead RNA) as detailed below:

(52) 1. For spin-column/spin-plate: 95% ethanol is added to the lysate/supernatant (1:1 ratio) and the mixture is loaded into the column. Column is then washed by Direct-zol RNA PreWash and RNA Wash Buffer several times and RNA is eluted with DNase/RNase-Free Water.

(53) 2. For the silica magnetic bead format: Direct-zol Binding Buffer is added to the lysate/supernatant (1:1 ratio) and mixed. Magnetic silica beads (MagBinding Beads) are then added to the mixture to bind nucleic acids. Beads are washed by 95-100% ethanol, Direct-zol MagBead PreWash, and 95-100% ethanol again, several times. During the wash steps, DNase I treatment can be performed with provided DNase I/10 Reaction Buffer. Beads are dried and DNase/RNase-Free Water is added to elute the RNA or DNA/RNA.

(54) Results of the whole blood extraction are shown in FIG. 6 and demonstrate highly efficient isolation of RNA and DNA from the samples.

Example 6: Selective Isolation of Small RNAs

(55) Studies were undertaken to determine if small RNA molecules could be selectively enriched using the methods of the embodiments. An example protocol for such selective enrichment is shown below:

(56) 1. Human epithelial cells were lysed in TRI Reagent and cleared by centrifugation.

(57) 2. 95% ethanol is then added to the sample lysate (1:1 ratio) and the mixture is loaded into the column.

(58) 3. small-RNA Elution Buffer, which comprises at least one chaotropic salt and at least one alcohol was added to the column. This removes/elutes small RNAs from the column (<200 nt).

(59) 4. To the flow-through that contains the small-RNAs (<200 nt), add 1 volume 95% ethanol and load the mixture into a new column.

(60) 5. Column is then washed by Direct-zol RNA PreWash and RNA Wash Buffer (see above) several times and RNA is eluted with DNase/RNase-Free Water.

(61) Results of these studies demonstrate that small RNAs could be selectively purified relative to larger mRNA and rRNA components.

Example 7: Isolation of RNA from Human Cells from Tri-Reagent with Different Binding Buffers

(62) RNA was isolated from about 50,000 human cells resuspended in 25 l water. All samples were lysed in 100 ml TRI Reagent and further agents were added as follows:

(63) (1) One volume (125 l) of either 100% methanol, ethanol, isopropanol, n-butanol, 3-methyl-1-butanol or a mixture thereof (FIG. 7) or (2) one volume (125 l) of a chaotropic salt or a buffer containing chaotropic salt, followed by two volumes (i.e. 250 l) of 95% ethanol (FIG. 8).

(64) Each mixture was loaded onto a separate spin Column (Zymo-SpinIC, Zymo Research Corp.), centrifuged at 12,000g with flow through being discarded after. Each column was then processed according to the protocol for the Direct-zol RNA miniprep, from step 3 on (Cat. No. 82050, Zymo Research Corp.). RNA was eluted with 15 l DNAse/RNAse-free water. High-quality RNA is evident from all alcohols or Dioxane or chaotropic salts tested (FIG. 7 and FIG. 8, agarose gel electrophoresis and spectrophotometric analysis is shown below In Tables 3 and 4).

(65) TABLE-US-00003 TABLE 3 Results for RNA binding with a variety of alcohol-based buffers Binding buffer component ng/l 260/280 260/230 methanol 104.57 2.01 2.14 ethanol 130.51 2.02 2.19 isopropanol 132.02 2.02 2.26 n-butanol 75.48 2.04 2.21 3-methyl-1-butanol (IAA) 53.13 1.98 2.00 methanol/ethanol 50/50 108.99 2.01 2.12 isopropanol/ethanol 50/50 133.24 2.03 2.26 n-butanol/ethanol 50/50 136.75 2.03 2.23 IAAl/ethanol 50/50 145.40 2.02 2.25 1,4-Dioxane 126.07 2.02 2.2

(66) TABLE-US-00004 TABLE 4 Results for RNA binding with a variety of chaotropic salts Binding buffer component ng/l 260/280 260/230 Binding buffer with GTC 107.32 2.07 2.18 Binding buffer with NaI 112.04 2.05 1.28 GHCl (6M) 130.82 2.06 2.2 Urea (9M) 105.22 2.02 2.05 Thiourea (1.8M)) 106.44 2.05 1.81 NaI (7M) 87.17 2.02 2

REFERENCES

(67) Each of the foregoing documents is hereby incorporated by reference in its entirety:

(68) U.S. Pat. Nos. 4,843,155; 5,472,872; 6,210,945

(69) U.S. Patent Publication 20100222560

(70) PCT Patent Publn. WO 98/31840.

(71) Sambrook et al., In: Molecular CloningA Laboratory Manual, 2001.