GB2538955A - Improvements in and relating to extracting biological analytes - Google Patents

Abstract

This invention relates to a method for extracting a plurality of analytes (e.g. DNA, RNA or protein) from a single starting biological sample; wherein the starting sample is deposited onto a solid support (e.g. paper, card, fibrous cellulose), a portion of the solid support containing the biological sample is removed and multiple processing steps are performed on this portion to sequentially extract said plurality of analytes.

Description

Improvements in and relating to extracting biological analytes

FIELD OF THE INVENTION

The present invention relates to extracting and testing for different plural biological analytes on a 5 solid sample material support containing a biological material sample deposited thereon.

BACKGROUND OF THE INVENTION

Herein, 'biological material' includes but is not limited to bodily fluids and tissues, plant, fungal, bacterial, viral or other microbial matter. Also, herein 'biological sample' includes all, or an 10 aliquot of said biological material. Herein, analytes' means elements of interest which are part of or contained in the biological sample.

Methods for the determination of, and extraction of, plural analytes such as nucleic acids and protein molecules from a biological sample has been carried out previously, for example as 15 proposed in US2009/0143570. Such methods have the advantage that the presence of plural analytes can be determined for example as biomarkers for certain diseases.

The determination of single analytes has also been made using samples deposited on solid supports, such as paper or other fibre based supports. These solid supports have an advantage that when the sample is dried they will inhibit its degradation. In addition, chemical coatings can be added to the solid support which will better preserve the sample, adding to the chances of successfully analysing the constituent of interest contained in the sample when there is a time delay between sample collection and analysis. To this end, different assays have been developed which can be used to determine the presence of a specific analyte deposited as a sample on a solid support with or without a chemical coating, but these assays each require a portion of the solid support to be removed from the solid support, each portion being used for a specific assay. Where the quantity of sample is very small, using different portions of the sample for each assay can be impossible. Where the sample is infectious, using different portions for each assay can be hazardous. In all cases, sequentially taking portions of the sample, and performing different assays is time consuming.

In addition, where different sample portions are taken, there is no guarantee that each portion will have the same characteristics as other portions. For example, where the ratio of the concentrations of two analytes is important, say in clinical diagnosis, it will add to the uncertainty of diagnosis to take two portions of the sample because these two portions may have been exposed to slightly different environmental conditions, and therefore the ratio sought may not be a true reflection of the original ratio.

The inventors have devised a way that a single sample on a solid support, or a single portion of a solid support, containing a biological sample can be used to determine the presence of plural analytes, for example DNA, RNA and/or protein molecules without the need to take multiple starting samples, where all types of DNA, RNA and proteins, are considered herein to be three different analytes.

SUMMARY OF THE INVENTION

The invention provides a method according to claim 1 having preferred features defined by claims dependent on claim 1. The invention provides also an assay kit.

The invention extends to any combination of features disclosed herein, whether or not such a combination is mentioned explicitly herein. Further, where two or more features are mentioned in 20 combination, it is intended that such features may be claimed separately without extending the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be put into effect in numerous ways, illustrative embodiments of which are 25 described below with reference to the drawings, wherein: Figure I shows gDNA isolated from HeLa cells by a first method according to the invention; Figure 2 shows gDNA isolated from Rat Liver by a first method according to the invention; Figure 3 shows results of PCR Amplification of gDNA isolated using f3-Globin primers using a first method according to the invention; Figure 4 shows results of PCR Amplification of gDNA isolated using P53 primers using a first method according to the invention; Figure 5 shows results of RNA isolation from HeLa cells using a first method according to the invention; Figure 6 shows the results of RNA isolated from HeLa cells using an Agilent Bioanlyzer using a first method according to the invention; Figure 7 shows bands of proteins extracted from rat liver using a first method according to the invention; Figure 8 shows the recovery of exogenously-added IL-2 from dried blood spots applied to various paper matrices using a second method according to the invention; Figure 9 shows the recovery of exogenously added IL-2 from dried blood spots applied to paper 10 coated with various chemicals using a second method according to the invention.

Figure 10 shows the recovery of exogenously-added IL-2 from dried blood spots applied to papers with paired combinations of chemicals using a second method according to the invention.

Figure 11 shows rat liver gDNA end-point PCR where the gDNA is obtained using a second method according to the invention; Figure 12 shows ciPCR results and amplification plot of gDNA derived from HeLa cells (0.069-69Ong DNA) purified using a second method according to the invention; Figure 13 shows an amplification plot of RT-ciPCR where RNA was derived from rat liver and purified using a second method according to the invention. The data shows RT-PCR products derived from 18s RNA, P450 RNA and the cFOS amplified from total RNA; Figure 14 shows an amplification plot of RT-qPCR, where RNA was derived from various rat tissues purified using a second method according to the invention. The data shows a RT-PCR product derived from cFOS amplified from total RNA; and Figure 15 shows direct measurement of interleukin from a solid support, purified using a third method according to the invention.

The invention, together with its objects and the advantages thereof, may be understood better by reference to the following description taken in conjunction with the accompanying drawings.

The methods described herein start with collecting a biological sample by depositing biological 30 material onto a solid support matrix. In the examples below, the samples may be deposited on a solid support matrix in the form of uncoated cellulose fibre material, such as paper. However, other solid support materials and their chemical coatings are suggested below. It is probable that the sample will be allowed to dry on the solid support, for example by storing the solid support in a dry environment. This is particularly advantageous when the sample is taken remotely, and its subsequent analysis, for example, according the methods outlined below, is performed in a different location, perhaps some time after the sample is taken. Herein, the term dry, or drying, means to allow or cause a majority of the liquid phase of a sample to evaporate.

In the methods described below, the dried sample is processed such that different analytes, for example genomic DNA, RNA and proteins are extracted and their presence as analytes can then be determined. In this case the presence of the analytes can be used, for example to diagnose a disorder. Obtaining the analytes is, in each method, carried out by using the whole sample on the solid support, or more likely, a single portion of the solid support containing an aliquot of the sample, such that just a single starting point of biological sample is required, and multiple handling of the biological material is not required. This is particularly advantageous if the biological material remains highly infectious for some time after the material is deposited onto the solid support.

METHOD 1 The separation and purification of nucleic acids and proteins from a single undivided biological 20 sample from a paper support using a spin column which includes a silica membrane.

This method allows the isolation of native protein, RNA and DNA from a single sample applied to a paper material.

Table 1-Buffers, Solutions and Columns Used

Description Composition

Lysis buffer 7M Guanidine HCI, 50mM Tris, 2% Tween 20, pH 7 (13-Mercaptoethanol added to 1%).

Wash Buffer 10mM Tris, 1mM EDTA, pH 8.0 (before use, 4 parts of ethanol added to 1 part of buffer).

Genomic DNA elution buffer 10mM Tris, 0.5mM EDTA, pH 8.0 RNA elution buffer Water Mini Column Silica membrane spin column (GE Healthcare) -1\ steps Homogenization The plain paper (for example as sold by Whatman under the trade name 903) containing a biological sample containing a sample of DNA, RNA and protein was added to a suitably sized tube for homogenization of a small volume of sample (i.e.350M or more). 3500 of lysis buffer containing 2-mercaptoethanol was added to the tube. The paper holding the sample was homogenised in a homogenizer according to instrument's user manual. The prepared homogenate was visually inspected to ensure thorough disassociation of the paper fibres, with no paper pieces visible.

DNA Purification and Binding A spin column was placed into a 2 ml collection tube. The above mentioned homogenate was 15 transferred into the spin column and spun for 1 minute at 11 000 x g. The flow-through was saved for RNA and Protein isolation at room temperature. The column and its remaining contents were transferred to a new 2 ml collection tube.

Column Wash 500u1 of lysis buffer was added to the column. The column was spun at 11000xg for 1 minute. The flow-through was discarded. The column was placed back into the same collection tube. 5000 of wash buffer was added to the column. The column was spun again at 11000xg for 1 minute. The column was transferred to a DNA-free 1.5m1 micro centrifuge tube.

DNA Elution 1001.11 of gDNA elution buffer was added to the centre of the column. The column was spun in a centrifuge at 8000xg for 1 minute. The column was discarded and the flow-through containing pure DNA was stored in the tube at minus 20°C. Figures I to 4 illustrate experimental results of various gDNA recovered using the method steps mentioned above and subjected to known amplification and electrophoresis separation techniques.

RNA Purification and binding 3500 of 100% acetone was added to the RNA/protein flow-through from the step above to form a solution. The solution was well mixed by pipetting up and down several times. A new spin column was placed in a new collection tube. The entire solution was transferred to the new column. The solution was spun in a centrifuge at 11000xg for 1 minute. The flow-through was saved for protein purification. The column was transferred to a new 2m1 collection tube.

Column Wash 500u1 of wash buffer was added to the column. The Column was spun in a centrifuge at 1000xg 20 for I minute. The column was transferred to an RNase free I.5m1 micro centrifuge tube.

RNA Elution 100u1 of RNA elution buffer was added to the centre of the column. The column was spun in a centrifuge at 8000xg for I minute. The column was discarded and the flow-through containing pure RNA was stored at minus 80°C until needed. Figures 5 and 6 illustrate experimental results of various RNA recovered using the method steps mentioned above and subjected to known amplification and electrophoresis separation techniques.

Total Protein Purification usinu 2-D Clean-Up Kit (GE Healthcare) 30 All protein precipitation steps below were carried out on ice.

Protein Precipitation The protein flow-through liquid obtained from the steps above was used for the protein precipitation. The liquid was well mixed and l00µ1 was transferred to a new 1.5ml micro-centrifuge tube. 3000 of precipitant was added to the liquid and mixed well. The tube was 5 incubated on ice for 15 minutes. 300p1 of Co-Precipitant was added to the liquid and mixed. The tube was spun in a centrifuge at maximum speed for 5 minutes. The supernatant was removed by pipetting (although decanting would also be suitable) as completely as possible. 441 of Co-precipitant was added on top of the protein pellet remaining after pipetting and was incubated on ice for 5 minutes. The tube was spun in a centrifuge at a maximum speed for 5 minutes. The 10 supernatant was again removed carefully and discarded.

Protein Pellet Wash 250 of deionized water was pipetted onto the pellet. The tube containing the pellet was mixed in a vortex machine for 5 minutes. 1m1 of pre-chilled wash buffer and Sul of wash additive was added to the tube. The tube was again mixed vigorously in a vortex machine. The tube was: incubated at minus 20°C for 30 minutes, whilst being mixed in a vortex machine for 30 seconds once every 10 minutes. The tube was spun in a centrifuge at a maximum speed for 5 minutes. The supernatant was carefully removed and discarded. 100p1 of 5% SDS (7M urea could be used also) was added to the protein pellet in the tube and mixed vigorously to dissolve the pellet. The tip of a pipette was used to break up the pellet initially. The tube was incubated for 3 minutes at 95°C to completely dissolve and denature the protein. The tube and contents were then cooled at room temperature. The supernatant containing the protein was then processed by known techniques i.e. SDS-PAGE and Western Blotting to determine the presence of predetermined proteins. Figure 7 illustrates experimental results for obtaining proteins using the method steps mentioned above and subjecting the proteins to known electrophoresis separation techniques. The purified proteins can be stored at -20°C for further processing.

METHOD 2 Preparation of nucleic acids and proteins from a single undivided sample using chemically coated or chemically impregnated paper.

In this method, satisfactory results may be obtained using papers impregnated with i) a mild non-ionic detergent such as NP40, Triton X-100, CHAPS to allow cell lysis without the subsequent denaturation of proteins; and/or ii) polyvinylalcohol (PVA), polyethyloxazoline (PEOX), poly(vinylpyrrolidone)(PVP) and poly(ethyleimine) (PEI), polyethylene glycol (PEG), each to minimise/prevent the absorption of 10 proteins thereby facilitating their elution.

Method steps Recombinant IL-2 ± carrier (R & D Systems; Cat. 202-IL-CF-10µg; lot AE4309112 and Cat. 202IL-10µg; lot AE4309081 respectively) was dissolved in Dulbecco's PBS without calcium and 15 magnesium (PAA; Cat. H15-002, lot H00208-0673), EDTA-anti-coagulated human, rabbit and horse blood (TCS Biosciences) at 50 pg or 100 pg/R1.

Aliquots (1 RI containing 0, 50 or 100 pg of IL-2) were applied to the following GE Healthcare filter papers; 903 Neonatal STD card, Cat. 10538069, lot 6833909 W082; DMPK-A card, Cat.

WBI29241, lot FT6847509; DMPK-B card, Cat. WB I 29242, Lot FE6847609 and DMPK-C card, Cat. WB129243, Lot FE6847009. Samples were allowed to dry overnight at ambient temperature and humidity.

Punches (3 mm diameter) were extracted from each paper type using the appropriately sized Harris 25 Uni-core punch (Sigma, Cat.Z708860-25ea, lot 3 I I 0). Single punches were placed into individual wells of the IL-2 microplate derived from the Human IL-2 Quantikine ELIS A (R & D Systems, Cat. D0250, lot 273275).

These plates were coated with a mouse monoclonal antibody against IL-2. The IL-2 protein was 30 eluted from the paper punch using the assay buffer (100 RI) supplied with the Quantikine kit. All subsequent steps were performed according to the instructions supplied with the Quantikine kit using a "paper in" method (paper punches are placed directly into the assay buffer and the analyte eluted directly in situ). On completion of the assay the optical density of the microplate was monitored at 450 nm using a Thermo Electron Corporation, Multiskan Ascent. The recovery of IL-2 was determined by comparing values to a standard curve of known 1L-2 concentrations. A fresh IL-2 standard curve was prepared for each individual experiment.

Additional experiments involved the addition of 1L-2-spiked blood to 903 Neonatal STD cards after the cards had been saturation dipped in several chemical solutions (as described below). In certain instances the paper was also saturation dipped in a mixed solution containing several of these chemicals to determine if the chemicals exhibited any additive or synergistic effect on the recovery of IL-2 from the dried blood spots.

Table 2a Paper coating chemicals used: A list of the chemicals and their sources is given below.

Poly-vinyl-alcohol (Sigma; Cat. P8136, lot 039k0147).

Poly-ethyl-enemine, 50% in water (Fluka; Cat. P3143, lot 29k1492).

Poly-vinyl-pyrolodine, 1% in water (Sigma; Cat.PVE40-100 mg, lot 11pk0097).

Inulin, 1% in water (Sigma; Cat. 12255-100 g, lot 079F7110).

Poly-2-ethyl-2-oxazoline, 1 % in water (Aldrich Cat. 372846, lot 30498PJ).

Tween 20, 1% in water (Sigma, Cat. P7949-100 ml, lot. 109k01021).

a-f3-Trehalose, 10 mg/m1 (Sigma, Cat. T0299-50 mg, lot 128k1337).

Albumin, 1% in water (Sigma, Cat A2153-10 g, lot 049k1586).

Caes n from bovine milk, 1% in water (Sigma, Cat. C5890-500 g, lot 089k0179).

Poly-ethylene glycol 1000, 1% in water (Biochemika, Cat. 81189, lot 1198969).

Poly-ethylene glycol 200, 1% in water (Fluka, Cat. 81150, lot 1384550).

The chemicals in Table 2a were used as a soak for the papers used. Following soaking, the papers were dried at ambient temperature & stored desiccated before use in the above method 2.

In an additional series of experiments DNA and RNA was isolated from the solid supports using the following procedure: Nucleic Acid (DNA & RNA) Elution from the Solid Support Table 2b Buffers, Solutions and Columns Used

Description Composition

Lysis buffer 7M Guanidine HCI, 50mM Tris, 2% Tween 20, pH 7 (P-Mercaptoethanol added to 1%).

Wash Buffer 10mM Tris, 1mM EDTA, pH 8.0 (before use, 4 parts of ethanol added to 1 part of buffer).

Genomic DNA elution buffer 10mM Tris, 0.5mM EDTA, pH 8.0 RNA elution buffer Water Mini Column Silica membrane spin column (GE Healthcare) Homogenization Add the solid support containing the sample of DNA, RNA to a test tube. Select a suitable sized tube for homogenization of a small volume of sample (i.e.350p1 or more). Add 350111 of lysis buffer containing 2-mercaptoethanol to the tube. Homogenize the solid support containing the sample on a homgenizer according to instrument's user manual. The prepared homogenate was visually inspected to ensure thorough disassociation (homogenization-with no solid support pieces should be visible).

DNA Purification and binding Place a new mini column into a 2 ml collection tube. Transfer the homogenized solid support 15 (3500) to the silica based spin column. Spin for I minute at 1 1 000 x g. Save the flow through for RNA isolation at room temperature. Transfer the column to a new 2 ml collection tube.

Column Wash Add 500p1 of lysis buffer to the column. Centrifuge at 11000xg for 1 minute. Discard the 20 flowthrough. Place the column back into the same collection tube. Add 500p1 of wash buffer to the column. Centrifuge at 11000xg for 1 minute. Transfer the column to a DNA-free 1.5m1 micro centrifuge tube.

DNA Elution Add 100µl of gDNA elution buffer to the centre of the column. Centrifuge at 8000xg for I minute. Discard the column and store the tube containing pure DNA at -20°C.

Total RNA purification and binding Place a new spin column in a new collection tube. Add 350111 of 100% acetone to the RNA/protein flow-through from the step above. Mix well by pipetting up and down several times. Transfer the entire mixture to the column. Centrifuge at 11000xg for I minute. Transfer the column to a new 10 2m1 collection tube.

Column Wash Add 5000 of wash buffer to the column. Centrifuge at 1000xg for 1 minute. Transfer the column to an RNase free I. 5m1 micro-centrifuge tube.

RNA Elution Add 1000 of RNA elution buffer to the centre of the column. Centrifuge at 8000xg for 1 minute. Discard the column and store the tube containing pure RNA at -80°C until needed.

Experimental Results When IL-2 was dissolved in EDTA-anti-coagulated blood, the 903 and DMPK-C cards facilitated the recovery of 45 -55% of the cytokine, while only 2 -3 % was recovered from the DMPK-A and B cards (see Table 1 and Figure 1). The 903 and DMPK-C cards are the basic base papers and have not been dipped or coated with any chemical, whilst the DMPK-A and B cards are coated with a proprietary mixture of chemicals that facilitate the denaturation and inactivation of proteins, micro-organisms and cells respectively. The DMPK-A and B cards have been designed to facilitate the storage of nucleic acids. Therefore the low IL-2 recovery levels observed when using the DMPK-A and B cards may actually be a reflection of the presence of these denaturing reagents and the ELISA-based antibody detection system used. The ELISA detection system requires the eluted IL-2 to exhibit an intact native structure.

Table 2c -shows the Recovery of exogenously-added IL-2 from dried blood spots applied to various paper types. The p-value compares ± carrier for each paper type. The presence of the carrier had no significant effect on the recovery of IL-2 (p-value >0.05) Paper Type W-2 recovery (%) p-value 903; minus carrier 46.9 ± 13.3 >0.05 903; plus carrier 50.7 ± 5.8 DMPK A * minus carrier 2.0 ± 0.0 >0.05 DMPK A; plus carrier 2.0 ± 0.0 DMPK B; minus carrier 2.0 ± 0.0 >0.05 DMPK B; plus carrier 2.0 ± 0.0 DMPK C; minus carrier 53.9 ± 4.8 >0.05 DMPK C; plus carrier 45.2 ± 5.4 No IL-2 recovery was observed when the cytokine was dissolved in phosphate buffered saline (PBS) irrespective of the paper type used (data not shown). The IL-2 recovery levels observed in the absence of added IL-2 were essentially equivalent to background levels indicating that the EDTA-anti-coagulated blood contain negligible amounts of endogenous IL-2 (data not shown).

Several chemicals were used to saturation dip the 903 Neonatal STD cards, some of which appeared to facilitate the recovery of elevated IL-2 levels compared to non-dipped papers (p-value < 0.05). For the 903 Neonatal STD cards (Table 2 and Figure 2), chemicals such as poly-vinylalcohol, poly-vinyl-pyrolodine, poly-2-ethyl-2-oxazoline, Tween 20, a-f3-trehalose, albumin and casein facilitated an IL-2 increased mean recovery > 55 % compared to -45% observed for the corresponding un-dipped paper.

Table 2d -The Recovery of exogenously-added IL-2 from dried blood spots applied to 903 Neonatal STD papers coated with various chemicals. The table is derived from 2 independent experiments (n = 6). The p-value compares the values derived from the dipped papers to those derived from the un-dipped 903 paper.

Chemical IL-2 recovery (%) p-value Un-dipped 44.9 ± 6.5 n/a Poly-vinyl-alcohol (PVA) 62.6 ± 11.2 < 0.05 Poly-ethyl-enemine (PET) 41.8 ± 6.0 > 0.05 Poly-vinyl-pyrolodine (PVP) 62.0 ± 10.9 < 0.05 Inulin 50.4 ± 7.6 > 0.05 Pol y-2-ethyl -2 -oxazol in e (PeOX) 66.1 ± 12.6 < 0.05 Tween 20 67.1 ± 9.0 < 0.05 u-f3-Trehalose 54.8 ± 8.6 < 0.05 Albumin 73.8 ± 13.6 <0.05 Caes n 55.0 ± 7.8 < 0.05 Poly-ethylene glycol 1000 42.5 ± 9.1 > 0.05 (PEG 1000) Poly-ethylene glycol 200 43.3 ± 11.0 > 0.05 (PEG 200) The saturation dipping of 903 Neonatal STD papers with a combination of two different chemicals indicated an additive effect in terms of the IL-2 recovery levels. For example, Table 2 demonstrates that the recovery of 903 Neonatal STD papers coated with Tween 20 and Albumin are 67 % and 74 % respectively. These figures are 22% and 29% greater than the equivalent un-dipped 903 paper respectively. Table 3 shows the cytokine recovery when dried blood spots containing exogenously added 1L-2 are applied to 903 Neonatal STD paper co-dipped with both chemicals. The recovery value for the Tween 20/Albumin coated paper is 92 % which represents an increase of 40% compared to the corresponding un-dipped paper.

Significantly increased IL-2 recoveries (p-value < 0.05) were observed when the 903 paper was co-dipped with the following chemical combinations, Poly-vinyl-pyrolodine (PVP) & Tween 20; Poly-vinyl-pyrolodine (PVP) & Albumin; Tween 20 & Albumin; Poly-2-ethyl-2-oxazoline (PeOX) & Tween 20; Poly-2-ethyl-2-oxazoline (PeOX) & Albumin; Poly-ethyl-enemine (PEI) & Tween 20 and Poly-ethyl-enemine (PEI) & Albumin.

Table 2e -The Recovery of exogenously added IL-2 from dried blood spots applied to 903 Neonatal STD papers coated with paired combinations of chemicals (n = 4).

Chemical IL-2 recovery (%) p-value Un-dipped 50.7 + 4.9 ilia Poly-vinyl-pyrolodine (PVP) & 48.1 + 13.4 > 0.05 Poly-2-ethyl-2-oxazoline (PeOX) Poly-vinyl-pyrolodine (PVP) & 79.4 + 18.8 < 0.05 Tween 20 Poly-vinyl-pyrolodine (PVP) & 70.5 + 13.2 < 0.05 Albumin Tween 20 & Albumin 92.0 + 11.0 < 0.05 Poly-2-ethyl-2-oxazoline (PeOX) & Tween 20 80.9 + 21.1 < 0.05 Poly-2-ethyl-2-oxazoline (PeOX) & Albumin 89.3 + 18.9 < 0.05 Poly-2-ethyl-2-oxazoline (PeOX) & Poly-ethyl-enemine (PEI) 61.8 + 15.8 > 0.05 Poly-ethyl-enemine (PEI) & 66.8 + 6.2 < 0.05 Tween 20 Poly-ethyl-enemine (PEI) & 75.8 + 13.1 < 0.05 Albumin Poly-vinyl-pyrolodine (PVP) & 56.4 + 11.0 > 0.05 Poly-ethyl-enemine (PET) METHOD 3 Isolation of DNA, RNA and proteins from biological samples applied to sample collection cards using direct chemical methods The protocol below describes methods that when combined facilitate the sequential isolation of proteins and nucleic acids. RNA and DNA can be separated from a solution of total nucleic acid by the use of selective precipitation agents.

Method steps A biological sample was applied to sample collection cards consisting of a cellulose-based solid support. The cards used included DMPK-A, -B and -C (GE Healthcare). DMPK-B and -C are cards that are impregnated with a chaotrope in the form guanidine thiocyanate, or a weak base, a chelating agent and an anionic surfactant such as SDS, and optionally uric acid or a uric acid salt, that confer properties such as efficient cellular lysis, prolonged storage of analytes such as DNA etc. DMPK-A is an example of a card that lacks a chemical coating (or impregnation). The sample was allowed to dry for at least 3 hours at ambient temperature. After which the cards were sealed and stored in an air tight bag containing desiccant. When ready to use, the cards were removed from the protective storage package and multiple 3 mm diameter discs containing the biological sample were excised from the solid support using a disposable Harris 3 mm Uni-core punch.

Subsequent protein and nucleic acid isolation was performed by combining multiple discs.

Protein extraction The excised discs were added to reaction tubes and protein elution buffer comprising phosphate buffered saline pH 7.2 and 0.05% Tween 20 was added. To facilitate protein elution, the tubes were shaken for 2 hours at either 4°C or ambient temperature. The eluted proteins were subsequently transferred to micro-titre wells pre-coated with monoclonal antibody raised against ferritin. The punches were retained and subsequently used to isolate total nucleic acid.

To assess the efficacy of the protein elution method recombinant IL-2 ± carrier (R & D Systems; 30 Cat. 202-1L-CF-10m; lot AE4309112 and Cat. 202-1L-10pg; lot AE4309081 respectively) was dissolved in blood (TCS Biosciences) at 50 pg or 100 pg/gl. Aliquots (1 pI containing, 50 (B) or (A) pg of IL-2) were applied to sample collection cards. These samples were allowed to dry overnight at ambient temperature and humidity. 3mm diameter punched disks were extracted from each paper type using the appropriately sized punch. Single discs were directly analysed for IL-2 with reagents from a fully configured IL-2 Quantikine ELISA kit (R & D Systems, Cat. D2050, lot 273275). Direct assays were carried out "punch in well", i.e., where a portion of the 903 filter paper was punched out and deposited in a reaction well of a convention multiwall plate.

On completion of the assay the optical density was monitored at 450 nm. The recovery of IL-2 was determined by comparing values to a standard curve of known IL-2 concentrations. Recovery rates are shown in Figure 15, and demonstrate that effective amounts of a protein can be recovered when the protein is deposited on a solid support. Thus, a protein from a biological sample such as blood is stable on a solid support and may be eluted and quantified using immunology methods such as ELISA.

The above protein elution method has also been validated by Cook et al 1998, (Blood 92, 1807- 1813), measured the H-and L-forms of ferritin using an ELISA in combination with monoclonal antibodies. The assay of L-ferritin was accomplished by adding 200 1"1.1 of eluted protein to duplicate micro-titre wells pre-coated with monoclonal antibody in 0.2 M carbonate buffer at pH 9.6 and incubating for 2 hours at room temperature. After washing the wells with PBS-Tween, 200 R1 of horse radish peroxidase -conjugated to anti-ferritin (or transferrin receptor) monoclonal antibody was added. After an additional, 2-hour incubation at room temperature, the wells were re-washed with PBS-Tween, and 200 R1 of ophenylenediamine dihydrochloride substrate in citrate-phosphate buffer pH 5.0 was added. After 30 minutes, the colour reaction was terminated by adding 50 pl of 2.5M sulphuric acid. The optical density was measured at 492 nm in a micro-plate reader and the ferritin values in the protein samples were determined from a standard curve of known concentrations. The protein elution efficiency from dried blood spots was defined as the ratio of ferritin in the dried blood spot to the level detected in whole blood. From the 10 samples analysed this ratio ranged from 0.93 -0.97 (mean 0.95 ± 0.01) indicating that a good recovery of protein is achievable using this method.

Nucleic acid extraction To reduce the effect of nucleases all equipment such as mortar, pestle etc. are pre-chilled in liquid nitrogen prior to use. Multiple 3 mm punches (up to 0.25 -2g weight) from the dried sample collection card/biological sample were removed using a Harris Uni-core punch and ground to a fine powder in liquid nitrogen. The cellulose-based powder was transferred to a 15 ml Falcon tube containing 4 ml of RNA/DNA extraction buffer (250 mM Iris-CI pH 8.5, 375 mM NaCI, 25 mM EDTA, 1% SDS and 1 % b-mercaptoethanol) and vortexed immediately. After which 3 ml phenol was added followed by 3 ml chloroform/isoamyl alcohol (24:1 v/v), the mixture was vortexed 15 sec. after each addition. The Falcon tube was centrifuge 10 min at 5000 rpm (-3000 x g) in Beckman JA-20 rotor. The aqueous phase (-6m1) was transferred to a new 15 ml Flacon tube. The phenol/chloroform (1:1) extraction and centrifugation step was repeated. Finally, to precipitate total nucleic acids, the aqueous phase (-6m1) was transferred to a new 15 ml Falcon tube containing 8 ml isopropanol. The solution was mixed and stored at -20°C overnight.

After cooling, the nucleic acids (RNA and DNA) were pelleted by centrifugation at 7000 rpm (-6000 x g), -30 minutes, and 4°C in Beckman centrifuge JA-20 rotor. The supernatant was decanted and the tubes drained by inverting on paper towel. The resultant nucleic acid pellet was re-suspended in 500 pl DEPC-treated water. The solution was re-centrifuged at 3000 rpm 5 min to pellet insoluble material such as residual cellulose fibres.

RNA isolation -To isolate large RNA molecules the supernatant was transferred to an Eppendorf tube containing 500 pi 4M Lin, mixed and incubated overnight at 4oC. After which the tube was spun at top speed in microfuge, 15 minutes at room temperature. Small RNA, e.g. tRNAs, and DNA remain in the supernatant, however, these can be separated using procedures outlined below). The pellet was re suspended in 200 pl DEPC-treated water. If any residual cellulose was present an additional centrifugation step was performed. RNA concentration and purity was assessed using A260 and A280 spectrophotometric readings and by applying the RNA sample to a Bio-analyser. RNA quality was also assessed by subjecting a sample to agarose gel electrophoreses using ethidium bromide to visual the presence of ribosomal RNA bands (data not shown).

DNA isolation -To isolate the DNA, 500 pl of the supernatant generated above was transferred to 30 each of two 1.5 ml microfuge tubes. To precipitate the DNA ethanol (1 ml) was added and the mixture incubated on ice 5-15 minutes after which the tube was centrifuged at top speed. No additional sodium or ammonium acetate was required due to the high salt concentration. The resultant DNA pellet was washed with 70% ethanol dried and re-suspended using 1541 water. The DNA samples were combined into one tube and spun 2 minutes to remove insoluble material. The supernatant was transferred to a fresh tube and 150 pl of 7.5 M ammonium acetate was added, 5 followed by mixing, and the addition of 900 (II ethanol. This precipitates the DNA for a second time. The mixture was incubated on ice for 15 minutes, and spun in a micro-centrifuge to pellet DNA. The resultant pellet was washed with 70% ethanol, dried and re-suspended in I 00 pi water. Because there is a substantial amount of small RNA in these samples, DNA concentration was estimated by subjecting aliquots to agarose gel electrophoresis and comparing the ethidium10 bromide signal versus known standards (data not shown).

METHOD 4 Alternative DNA and RNA extraction method Protein extraction is carried out as for method 3.

An alternative approach for the isolation of genomic DNA and RNA from cellular lysates that employs the use of compaction agents such as spermidine and hexammine cobalt for the selective precipitation of DNA and RNA respectively. The chromosomal DNA can be precipitated and removed from a cellular lysate with an initial spermidine precipitation. Specific RNA molecules can be subsequently and selectively precipitated or fractionated with the addition of increasing amounts of hexammine cobalt.

Total nucleic acid derived from a biological sample was extracted from the sample collection cards as described above. The resultant solution contained genomic DNA and total RNA. The addition of 2.5 mM spermidine at pH 6.9 to the total nucleic acid solution followed by room temperature incubation (20°C) for an hour mediates the selective precipitation of genomic DNA. The genomic DNA was then separated from RNA following a centrifugation step (10 min, 15,000g at 4°C). The resultant DNA pellet was re-suspended in TE buffer for subsequent downstream use.

To fractionate the RNA species present in the total RNA, hexammine cobalt was used. The 30 supernatant from above i.e. treated with 2.5 mM spermidine and cleared of genomic DNA was aliquoted (500 pl) in microfuge tubes. To precipitate ribosomal RNAs, hexammine cobalt was added to the sample to a final concentration of 2 mM, followed by vortex ng for 1 min, incubation for 15 min at 4°C, and finally a centrifugation step (10 min, 15,000g at 4°C). Increasing the concentration to 3.5 mM resulted in the precipitation of the remaining total RNA. Increasing the hexammine cobalt to 8 mM facilitated the isolation of low molecular weight RNA fraction (< 500 bases) by precipitation. The resultant RNA pellets were subsequently re-suspended using DEPCtreated water.

To confirm the selective precipitation of RNA, using hexammine cobalt, RNA based on an artificial 5S ribosomal RNA was used ("aRNA") was used. This was precipitated using 2 mM hexammine cobalt and the resultant RNA pellet was re-suspended and analysed using agarose gel electrophoresis. The results indicated that the aRNA was observed in the supernatant associated with the other ribosomal RNA species (data not shown). A smaller b-Ribozyme was also precipitated from a spermidine-cleared lysate by first using a 2 mM hexammine cobalt precipitation to remove larger RNA molecules and then a second 8 mM (final concentration) hexammine cobalt precipitation to fractionate the smaller ribozyme RNA species.

The above methods may also be used with the RNA stabilisation medium (GE Healthcare) for the isolation of DNA, RNA and protein from a single sample using a combination of either direct purification methods or a combination of both direct methods and micro-spin columns. In this instance the RNA stabilisation medium functions as a solid support and includes a solid support material coated with a chaotrope, a reducing agent, a weak acid and an antioxidant.

Although embodiments have been described and illustrated, it will be apparent to the skilled addressee that additions, omissions and modifications are possible to those embodiments without departing from the scope of the invention claimed. For example various sample processing procedures are described above using generally accepted terminology, however equivalent procedures will be apparent to the skilled person, and so these equivalent procedures are intended to be encompassed herein where the generally accepted terminology is used. So, for example, where homogenisation is described, and related terms such as homogenate, it will be apparent that any disruption or disassociation of the solid support material could be employed of sufficient extent to enable the procedure to be carried out effectively, not necessarily to the extent that an entirely homogeneous end product is produced. Likewise, such terms as extracting, binding, washing, eluting, precipitating, etc. are all to be interpreted as conducting such actions such that a sufficient quantity of the analyte is affected, rather than an exhaustively carrying out such an action.

Claims (12)

1. CLAIMS1. A method for extracting plural analytes from a single starting biological sample, the method comprising the steps of a) depositing a biological material onto a solid support; b) taking a starting biological sample comprising at least a portion of the solid support containing at least an aliquot of said biological material deposited thereon; c) sequentially subjecting said starting sample to plural processing steps in order to extract said plural analytes.
2. 2. The method of any one of the preceding claims, wherein the solid support comprises: a fibrous material, for example cellulose fibres; or alginates; or fibrous polymeric materials
3. 3. The method according to any one of the preceding claims wherein said solid support is uncoated, or coated or impregnated with a chemical or chemical composition, selected from a group consisting of one or more of a weak base, a chelating agent and an anionic surfactant; a chaotrope, optionally including a reducing agent, a weak acid and an antioxidant; or coated or impregnated with a polymer, for example polyester, a vinyl polymer, a non-ionic synthetic polymer, in particular any one or more of the chemicals listed in the first columns of tables 2a, 2d or 2e.
4. 4. A method according to claim 1, 2 or 3, wherein said plural analytes include two or three of: DNA, RNA, and proteins.
5. 5. A method according to claim 4, wherein said plural processing steps include initially causing the elution of proteins off the starting sample of the solid support.
6. 6. A method according to claim 5, wherein said plural processing steps further include homogenising said starting sample in the presence of nucleic acid extraction buffer.
7. 7. A method according to claim 6, wherein said plural processing steps further include selective precipitation of DNA and/or RNA, optionally wherein said selective precipitation of DNA and/or RNA is in the presence of one or more of: polyethyleneglycol, isopropanol, ethanol, sodium acetate; ammonium acetate; glycogen; polyamine, such as spermidine and spermine; lithium chloride; hexammine cobalt; and calcium salts such as calcium chloride.
8. 8. A method according to claim 4, wherein said plural processing steps include homogenisation of said starting sample and passing the homogenate through a silica membrane to extract said DNA retained at said membrane.
9. 9. A method according to claim 8, wherein said plural processing steps further include collecting the liquid passed through said membrane and passing the collected liquid through a further silica membrane to extract said RNA retained at said membrane.
10. 10. A method according to claim 9, wherein said plural processing steps further include collecting the liquid passed through said thither membrane and extracting said proteins therefrom.
11. 11. A sample collection device including a solid material support when subjected to any one of the methods claimed in claims 1 to10.
12. 12. A kit including the sample collection device of claim 11, and reagents necessary for carrying out the method according to any one of claims 1 to 10.