Abstract:
A method for disrupting cells and/or tissues employs a disrupter with a disruption element having an outer dimension that is slightly smaller than the inside dimension of a container in which a sample is place to be disrupted. Devices for disrupting cells and/or tissues are also presented. The device and methods are used in processes such as the extraction of nucleic acids.

Description:
BACKGROUND  
       [0001]     RNA expression can be used as the basis of in vitro diagnostic tests useful in screening, diagnosis, prognosis, therapeutic monitoring, and/or follow-up of relapse of disease. Intact non-degraded RNA extracted from cells or tissues is important for the valid measurement of RNA expression. RNA expression is particularly useful in microarray and PCR assays involving numerous genes.  
         [0002]     In most RNA extraction methods, RNA is released from cells by mechanical means. Often, this is done in the presence of RNA stabilizing agents such as guanidium salt. This minimizes the degradation of RNA by RNAases released from the cell or tissue during the process. Devices for the mechanical disruption of biological samples are proposed in the following exemplary U.S. patents: U.S. Pat. No. 4,307,846 to Spelsberg; U.S. Pat. No. 5,197,483 to Rogalsy; U.S. Pat. No. 5,840,878 to Collis; U.S. Pat. No. 5,829,696 to DeStefano; U.S. Pat. No. 6,235,501 to Gautsch; and U.S. Pat. No. 5,567,050 to Zoblinsky. In such devices, an element is used to grind, pulverize, or otherwise contact the sample containing the cellular component that is to be extracted. Throughout this specification, this element is referred to as a disruption element. Most often the devices use a disruption element that is a bead or ball such as a glass bead.  
         [0003]     There is a continuing need to improve cell and tissue disruption methods so that RNA extraction can be made faster, more efficient, more productive, and amenable to automation. This is particularly desirable in the case of intra-operative assays.  
       SUMMARY OF THE INVENTION  
       [0004]     The invention is a method of disrupting cells and tissues. Preferably, the method involves the use of a mechanical device and is conducted in 45 seconds or less.  
         [0005]     Another aspect of the invention is a method of extracting RNA from a cell or tissue sample. Preferably, the sample is a lymph node tissue. In the method, cells and/or tissues are mechanically disrupted, preferably in 45 seconds or less. Cellular debris and other non-RNA materials such as air bubbles are then removed from the sample exposed to the disruption The RNA is then extracted from the sample.  
         [0006]     In yet another aspect of the invention, a device for disrupting cells and tissues includes a containment portion in which a disruption element is placed. The space of the outer surface of the disrupter (circumference in the case of a bead) is just slightly smaller than the inner dimension of the containment portion (diameter in the case of a tube). 
     
    
     DETAILED DESCRIPTION  
       [0007]     In the most preferred embodiment of the invention, a mechanical cell and tissue disrupter such as the “FASTPREP” instrument available from Qbiogene, Inc. of Carlsbad, Calif. is used. Such disrupters are described in U.S. Pat. No. 5,567,050 which is incorporated herein by reference. Preferably, the method of the invention employs 2.0 ml screw-capped cryovial tubes containing an “O-ring”, or their equivalent. Between about 30 mg and 400 mg of tissue are placed in the tubes along with a disrupter element. Most preferably, the disrupter elements are steel beads having a diameter of 6.35 mm such as those available from BioSpec Products of Bartelesville, Okla. To the tube, is added a volume of homogenization buffer (e.g., RLT buffer from Qiagen Corp.) that is about half of the tube or more. Preferably, about 1 ml of such buffer is added when using the 2.0 ml tube. The homogenzation buffer contains an RLT stabilizer, preferably a guanidium salt. The disrupter is then engaged for 45 seconds or less and the sample is centrifuged, preferably in a microfuge, to remove the cellular debris and air bubbles from the samples. The supernatant is then decanted. Preferably, the supernatant is transferred into a spin column and the column is treated with different wash buffers to purify RNA. Solvent-based RNA purification methods can be used in place of the spin column if desired too.  
         [0008]     The device of the instant invention is a disrupter such as one described above but the dimensions and/or composition of the disruption element are provided that rapidly and efficiently disrupt the tissue in a way not previously possible. As described above, the disruption element is the portion of the device that makes contact with the tissue. Preferably, this is a bead. The disruption element of the invention has an outer dimension that is just slightly smaller than the inner dimension of the container in which the sample is processed. In the preferred embodiment, the container is a tube such as the one described above with an inner diameter of 8.28 mm and the disruption element is a 6.35 mm steel bead. Other containers and disruption elements can also be used provided that their inner and outer dimensions are of similar proportions. The density of the bead also contributes to the improved performance of the inventive device. Preferably, the bead is made of stainless steel with a density of 8.03 g/cm 3  or greater although beads having a density above 7.0 can be used.  
       EXAMPLES  
     Example 1  
     Evaluation of Different Bead Sizes and Composition  
       [0009]     Different bead sizes and materials were evaluated for their ability to disrupt frozen pig lymph node tissue. Two ml screw-cap tubes (BioSpec Products, Bartlesville, Okla.) with an 8.28 mm inner diameter were used. Each tube was half-filled with beads except for tubes containing a 6.35 mm (0.25 inch) stainless steel beads, in which case a single bead was added per tube. Between 200 and 250 mg of frozen, pulverized pig lymph node tissue was added per tube. The tubes were filled with Buffer RLT (Qiagen, Inc, Valencia Calif.) and homogenized for 1, 3, or 5 min by means of a Mini-Bead Beater 8 instrument obtained from BioSpect Products).  
         [0010]     The following bead sizes and compositions were purchased from Biospec Products: 0.1 mm diameter zirconia/silica (cat 11107901 z), 0.1 mm diameter glass (cat 11079101), 0.5 mm diameter glass (cat 1107905), 0.5 mm diameter zirconia/silica (cat 11079105z), 1.0 mm diameter glass (cat 11079110), 2.5 mm diameter zirconia/silica (cat 11079125z), 6.35 mm diameter stainless steel (cat 11079635ss), and 6.35 mm diameter glass beads (110796325).  
         [0011]     Results of these studies are shown in Table 1 below and indicated best tissue disruption with multiple 6.35 mm diameter glass beads or a single 6.35 mm stainless steel bead, as determined visually with the following scale: (1) poorly homogenized tissue, (2) partially homogenized tissue, and (3) completely homogenized tissue.  
                                                                           TABLE 1                           Evaluation of Different Bead Size and Composition       on Node Tissue homogenization*                Node   Bead       Tissue                   tissue   size   Bead   Disruption       Tube no.   wt (mg)   (mm)   Composition   1 min   3 min   5 min                    1   207   0.1   zirconia/silica   1   1   1       2   221   0.1   glass   1   1   2       3   203   0.5   glass   1   1   1       4   204   0.5   zirconia/silica   1   1   1       5   225   1   glass   1   1   1       6   249   2.5   zirconia/silica   1   2   3       7   200   6.35   stainless   ND   2   3       8   230   6.35   glass   2   2   3                 Tissue disruption was determined visually as described in the text above.             
 
       Example 2  
     Effect of Bead Density  
       [0012]     Example 1 illustrates that complete tissue homogenization is best achieved using a disruption element (bead) of 6.35 mm in diameter. Bead density also affects the quality of homogenization.  
         [0013]     A comparison was made between beads possessing equal diameter (6.35 mm) but different densities. Approximately 225 mg of pig lymph node was disrupted in 2 ml screw-cap vials containing 1 ml of a guanidinium-based lysis buffer (Qiagen Buffer RLT), plus one 6.35 mm bead of ceramic, glass, or stainless steel. Tissue samples containing beads were processed for 1 minute, 3 minutes, and 5 minutes in the Biospec Bead Beater 8. Samples were briefly centrifuged and were assessed visually for quality of homogenization as in Example 1.  
                                                                                   TABLE 2                           Performance of 6.35 mm Beads of Various Densities on the       Homenization of Pig Lymph Node                Bead           Avg-   Tissue               Bead   Weight   Bead Density   Manufacturer/   Tissue   Disruption       size/type   (g)   mg/mm3   catalog #   Weight   1 min   3 min   5 min                    6.35 mm   276   2.74   Biospec/   248   1.00   1.67   2.00       glass           11079635       6.35 mm   679   6.75   Q Biogene cat.   243   2.33   3.00   3.00       ceramic           # 6910-052       6.35 mm   1008   10.02   Biospec/   226   2.67   3.00   3.00       stainless           11079635ss       steel                  
 
         [0014]     Table 2 summarizes the average of 3 replicates scored visually using the following scale: (1) poorly homogenized tissue, (2) partially homogenized tissue, and (3) completely homogenized tissue. This experiment shows that tissue disruption improves as the density of bead material increases, with the best performance achieved with stainless steel beads.  
       Example 4  
     RNA Yield and Purity with the BeadBeater Instrument and Glass or Stainless Beads  
       [0015]     RNA yield and purity were determined with either glass or stainless steel beads of several diameters of 6.35 mm or greater. The Mini-Bead Beater 8 was used at high speed for 1 min. Conditions were as described previously in example 1. Stainless steel and glass beads 6.35 mm were obtained from Biospec Products. All other beads were purchased from Glen Mills Inc, Clifton, N.J.  
         [0016]     After tissue disruption, the resultant homogenate was centrifuged 15 sec by means of an Eppendorf model 5415 (Brinkman Instruments, Westbury N.Y.), and RNA was purified by means a QIAGEN RNeasy Mini kit (Qiagen Inc, Valencia Calif.) A portion of the lysate was diluted to a concentration of 30 mg tissue/ml of Buffer RLT. Four hundred microliters of lysate was added to a microfuge tube containing 400 ul of 70% ethanol, and the sample was mixed by pipetting. Sample (700 ul) was applied to an RNeasy mini column placed on a vacuum manifold. Vacuum was applied and maintained during the remainder of the filtration steps. The column was washed with 700 ul of Buffer RWI, followed by 700 ul of Buffer RPE. The spin column was then placed in a 1.5 ml collection tube, and centrifuged at 14,000 RPM for 30 seconds to dry the column. The column was transferred to anew 1.5 ml collection tube. Fifty microliters of RNAase-free water was applied directly to the membrane, and the tube containing the column was centrifuged for 30 seconds at 14,000 RPM to elute the RNA. The RNA samples were placed on ice, and stored at −80C prior to assaying.  
         [0017]     RNA yield was measured spectrophotometrically based on the 260/280 nm ratio of the sample as determined on a Gene Spec III instrument (MiraiBio, Alameda Calif.). An Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Assay Reagents and Supplies (Agilent Technologies, Foster City, Calif.) were also used to determine RNA yield and quality.  
         [0018]     Results are summarized in Table 3, and demonstrate that a stainless steel bead 6.35 mm in diameter provides the highest RNA yield, and best tissue homogenization as determined by visual inspection of the tubes. Furthermore, a stainless steel bead, as compared to a glass bead of identical diameter, shows significant improvement in yield. Agilent Bioanalyzer electropherograms confirm that high quality RNA was obtained with the rapid homogenization method. Clearly defined 18 s and 28 s ribosomal peaks were shown in the samples disrupted by the rapid bead based method, and all samples had an 18 s to 28 s ratio greater than 1.5.  
                                                                   TABLE 3                           RNA Yield with Different Size and Composition*                                RNA yield           Node   Bead           ug RNA/       Tube   Tissue   diameter   Bead   Homogenization   mg       no.   wt (mg)   (mm)   Composition   1 min   tissue                    1   480   6.35   Stainless   4   0.069       2   400   6.35   Stainless   4   0.067       3   400   6.35   Stainless   3   0.073       4   392   6.35   Stainless   3   0.053       5   454   6.35   Stainless   4   0.085       6   456   6.35   Stainless   4   0.043       7   422   6.35   Glass   2   0.03       8   445   6.35   Glass   2   0.026       9   481   6.35   Glass   2   0.022       10   473   6.35   Glass   2   0.032       11   378   6.35   Glass   2   0.041       12   401   6.35   Glass   2   0.035       13   463   7   Stainless   4   0.018       14   503   8   Stainless   2   0.023       15   389   7.93   Stainless   2   0.027       16   537   7.14   Stainless   2   0.067                 *Tissue disruption was measured visually based on the following scale: 1-no homogenization, 2-poor homogenization, 3-partial homogenization (few pieces of tissue remain), 4-visible cell debris, and 5-complete homogenization.             
 
       Example 3  
     Tissue Disruption in the FastPrep Instrument  
       [0019]     The following experiment was performed to determine the precision of RNA yield for pig node samples weighing between 50 and 500 mg when disrupted in the FastPrep Homogenizer (Qbiogene, Inc, Carlsbad Calif.). These experiments were performed with 2 ml Sarstedt screw-cap vials containing a single 6.35 mm stainless steel bead.  
         [0020]     In these experiments, different weights of frozen and pulverized pig lymph node tissue shown in Table 4 were added into 2 ml Sarstedt screw-cap vials containing one 6.35 mm stainless steel bead per tube. One ml of Buffer RLT was added and the tubes were processed for 45 sec in a FastPrep instrument with a speed of 6.5 meters/sec. (To perform this experiment, the FastPrep instrument was modified by removing washers from the spokeplate so that this larger tube size could be accommodated). After disruption, tubes were then centrifuged at 14,000×g for 15 sec in an Eppendorf model 5415C (Brinkman Instruments, Westbury, N.Y.) to pellet cellular debris and remove air bubbles from the sample. A portion of each lysate was diluted to a concentration of 30 mg of tissue/ml of Buffer RLT, and mixed well by pipetting. The tissue was processed to extract RNA, and RNA was quantified in Example 3.  
                                                                                   TABLE 4                           Effect of Node Weight on RNA yield and Precision                    Tissue           Total RNA               Experimental       Weight   260/280   Total   mean       condition   Tube #   (mg)   ratio   RNA (ug)   recovery(ug)   SD   % CV                     50-100 mg   1   53   2.1   13.1   24.3   6.8   0.28       tissue   2   54   2.1   20.6           3   79   2.0   27.2           4   80   2.1   24.5           5   50   2.1   27.8           6   67   2.0   32.8       300-400 mg   19   357   2.0   23.7   22.2   1.3   0.06       tissue   20   359   2.0   22.1           21   383   2.0   20.2           22   357   2.1   22.1           23   367   2.0   23.7           24   325   2.1   21.6       400-500 mg   25   456   1.9   19.3   17.3   3.3   0.19       tissue   26   470   2.0   18.4           27   414   1.8   19.1           28   442   2.0   18.9           29   468   2.0   17.5           30   444   2.1   10.8                  
 
         [0021]     Results of these studies demonstrate total RNA recoveries of over 20 ug at samples less than 400 mg of tissue. Slightly less total RNA recovery was observed in pig node sample between 400 and 500 mg, suggesting slightly less efficient tissue disruption under these experimental conditions.  
         [0022]     Tubes containing tissue samples of lower weight had the highest imprecision However, in subsequent experiments, we have shown that higher standard deviations at lower node tissue weights are probably due to tissue heterogeneity and sampling since the precision of tissue recovery improves significantly when a larger tissue is pulverized and then a smaller tissue weight is used for homogenization.  
       Example 5  
     Realtime PCR Assay of Porcine B-actin Gene Expression  
       [0023]     The following experiment was performed to demonstrate that RNA recovered after rapid homogenization in the Fast Prep method is amplifiable in real-time PCR assays. Purified RNA from example 4 was used. For comparison, a control was included in which pig node tissue was disrupted by mechanical disruption by means of an Omni Tissue Homogenizer and disposable PCR probes (Omni International, Warrenton, Va.). The Omni tissue homogenizer does not use beads to enhance tissue disruption. For treatment in the Omni Tissue homogenizer, 100-400 mg of pig node tissue was mixed with 6 ml of Buffer RLT and homogenized with a hand homogenizer (model PCR258) for 1 min. Samples were mixed with an equal volume of 70% ethanol, and 700 ul was applied to a Qiagen spin column and RNA was purifed as described in Example 3.  
         [0024]     RNA was transcribed to make cDNA copies as follows: One μg of RNA was added to a master mix made by mixing 1.25 μl of a 75 μM stock solution of anchored Oligo dT23, 0.31 μl of 10 mM of each dNTP, and RNAase-treated water (Sigma Chemical Co, St. Louis, Mo.) to a volume of 13 μL, and the solution was heated to 70C for 5 min, and then placed on ice. Then a 9.5 μL mix comprising 5 μL of 5×Superscript First-Strand Buffer, 2.5 μL of 0.1M Dithiothreitol, 0.75 μL of a solution of 40 U/μL Rnasin (Promega Corp, Madison Wis.) and 1.25 μL of 200 U/μL Superscript II reverse transcriptase was added, and the tube was incubated at 42C for 50 min. Five μL of 0.5M NaOH was added, the tube was incubated at 70C for 5 min, and then Tris-HCI buffer, (2.5% of 1M, pH 7.0), was added, followed by 67.4 μl of RNAase free water. Assuming a complete conversion of RNA to cDNA, and a conversion factor of 1 ug equal to 50,000 cell equivalents (CE), a concentration of 500CE/μL in 25 mM Tris buffer was used for all subsequent real-time PCR assays.  
         [0025]     Porcine B-actin primers (SEQ ID NO. 1 and SEQ ID NO. 2) were synthesized by Invitrogen Corp. (Carlsbad, Calif.) and the B-actin Taqman probe (SEQ ID NO. 3) from SYNTHEGEN, LLC (Houston, Tex.). For all Taqman probes, carboxyfluorescein (FAM) and carboxytetramethylrhodamine TAMRA) were used as the dye and quencher pair.  
                                           SEQ ID NO. 1   CCACACGGTG CCCATCTACG                           SEQ ID NO. 2   AGGATCTTCA TGAGGTAGTC GGTCAG                       SEQ ID NO. 3   6-FAM -ACGCCCTGCC CCACGCCATC               CTGCGTCTGG-TAMRA          
 
         [0026]     For the Taqman assays, a master mix was prepared by adding 25 μl of 2×Taqman Universial PCR mix (Applied Biosystems, Inc), 1.25 μl of a 10 μM stock probe solution, and 5 μl of a 0.5 μM solution of each primer. cDNA (2 μl) was added to each optical reaction microtiter plate well. After capping with optical caps, the plates were processed in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Inc., Foster City, Calif.) and the assay was performed according to the protocol recommended by the manufacturer. An average of three determinations by Taqman assays are shown in Table 5.  
         [0027]     Results of these are summarized in Table 5, and demonstrate that comparable yields of RNA can be obtained with the method of this invention and the Omni Homogenizer. The method of the present invention is advantageous in that multiple samples can be processed simultaneously. Furthermore, since sealed tubes are used, sample to sample contamination is minimized.  
                                                                   TABLE 5                           Taqman Assay of RNA Homogenized by Omni Homogenizer       and by Fast Prep Instrument                    Avg                   Tissue Homogenization   Node Size (mg)   Ct   SD   % CV   Avg                    Omni Homogenizer   124   20.11   0.47   0.02               129   19.95   0.36   0.02           113   19.8   0.09   0           94   20.02   0.07   0   19.97           394   18.79   1.19   0.06           388   19.57   0.31   0.02           392   19.01   0.76   0.04           412   19.96   0.24   0.01   19.33       Fastprep Instrument   80   20.33   0.65   0.03           145   19.48   0.5   0.03           142   20.39   0.17   0.01           123   20.88   0.3   0.01   20.27           456   19.89   0.29   0.01           470   20.03   0.27   0.01           414   20.03   0.34   0.02           442   20.85   0.42   0.02   20.2                  
 
         [0028]