0001298B:4110133A.qxd 1/16/2009 2:40 PM Page COV2 Aurum™ Total RNA Fatty and Fibrous Tissue Kit Instruction Manual Catalog # 732-6830 The Aurum Total RNA Fatty and Fibrous Tissue Kit is composed of: Module 1 – 732-6870 (Aurum Total RNA Fatty and Fibrous Tissue Module) Module 2 – 732-6880 (PureZOL™ RNA Isolation Reagent, 50 ml), packaged and shipped separately For technical support, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723).
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10001298B:4110133A.qxd 1/16/2009 2:40 PM Page TOC1 Table of Contents Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Section 2 Kit Components . . . . . . . . . . . . . . . . . . . . . . . . .2 Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .3 Section 4 Materials and Equipment Required (Not Provided in the Kit) . . . . . . . . . . . . . . . . . . .3 Supplies for Tissue Grinding, Disruption, and Homogenization . . . . . . . . . . . . . . . .
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10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 1 Section 1 Introduction The Aurum™ total RNA fatty and fibrous tissue kit produces high yields of pure total RNA from samples that are difficult to disrupt. The kit is ideal for fatty or fibrous tissues, or samples that are rich in RNases. It also works well with most animal and plant tissues, cultured cells, yeast, and gram-negative and gram-positive bacteria. With this kit, greater than 100 µg of total RNA can be isolated from many sample types.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 2 Section 2 Kit Components The Aurum™ total RNA fatty and fibrous tissue kit contains the following components: Components* Quantity/Amount RNA binding mini columns Capless wash tubes, 2.0 ml Capped microcentrifuge tubes, 1.5 ml Capped microcentrifuge tubes, 2.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 3 Section 3 Storage Conditions Store components of the kit at the recommended temperatures (see Table 1 below). Table 1. Recommended storage temperature for the Aurum™ total RNA fatty and fibrous tissue kit components.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 4 Supplies for Tissue Grinding, Disruption, and Homogenization • Fresh tissue: tissue cutter • Frozen tissue: liquid nitrogen, mortar and pestle • Tissue homogenizer: rotor-stator homogenizers, or bead mill homogenizers recommended Additional Equipment Required for Vacuum Format • Aurum vacuum manifold with vacuum regulator and column adaptor plate (catalog # 732-6470), or other vacuum manifold with luer fittings.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 5 Section 5 Before Using the Aurum™ Total RNA Fatty and Fibrous Tissue Kit Please read the following guidelines before proceeding with the total RNA isolation. Maximum Starting Material Amounts* The Aurum total RNA fatty and fibrous tissue kit is designed to process up to the amounts indicated below (per column): • 1 x 107 cultured mammalian cells grown in suspension • One 102 cm plate mammalian cultured cells grown in monolayer • 2.
001298B:4110133A.qxd 1/16/2009 2:40 PM Page 6 yeast for processing. To calculate the volume of culture required, use the following equation: (OD600 of undiluted culture) x (culture volume in ml) = # OD•ml For example, 3 OD•ml of yeast would require 500 µl of an undiluted culture with an OD600 = 6. Note: 1 OD600 is equivalent to approximately 8 x 108 bacterial cells/ml, or 1 x 107 yeast cells/ml. Table 2.
10001298B:4110133A.qxd g 1/16/2009 2:40 PM Page 7 Reagents Used With the Aurum Total RNA Fatty and Fibrous Tissue Kit PureZOL™ RNA Isolation Reagent for Sample Lysis an Use 1 ml of PureZOL for up to: • 100 mg of tissue • 1 x 107 cultured cells grown in suspension • One 10 cm2 plate of cultured cells grown in monolayer • 2.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 8 (DEPC) to inactivate RNases. DEPC is an efficient, strong, and nonspecific RNase inhibitor that is usually used at a concentration of 0.1% • To prepare a 0.1% (v/v) solution of DEPC-treated water, add 1.0 ml of liquid DEPC per 1 L of water. Incubate the solution at 37°C for 1 hr while mixing thoroughly. Autoclave the treated water to remove the DEPC • Warning: DEPC is suspected to be a carcinogen and should be handled with care.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 9 • Nondisposable, nonautoclavable plasticware should be rinsed with 0.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 10 Table 3. Disruption and homogenization methods.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 11 Section 6 Vacuum Manifold Setup and Use With the Column Adaptor Plate Guidelines for Vacuum Format • The recommended operating range is –17 to –20 inHg. Do not exceed –25 inHg when performing this protocol. A vacuum regulator is strongly recommended to establish the appropriate negative pressure. Table 4. Pressure unit conversions. To convert from inches of mercury (inHg) to: millimeters of mercury or torr (mmHg, torr) millibar (mbar) Multiply by: 25.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 12 Vacuum Setup (Figure 1) 1. Cut tubing into three pieces of appropriate length. 2. Use one piece of tubing to connect to the right side of the vacuum regulator to the vacuum source. 3. Use another piece of tubing to connect the left side of the vacuum regulator to the sidearm of the filter flask. 4. Place a rubber stopper with hole into the mouth of the filter flask. Insert a serological pipette (or comparable) into the hole of the stopper. 5.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 13 Manifold Wash Setup (Figure 2) 1. Insert the CAP (luer ends up) into the depression in the vacuum manifold top. Ensure that the CAP rests evenly on the gasket. 2. Insert the luer ends of the desired columns into the available luer fittings, ensuring a tight fit. 3. Close the unused luer fittings with the caps provided. Close caps by rotating clockwise until light resistance is encountered.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 14 Section 7 Vacuum Protocol All steps are carried out at room temperature unless otherwise indicated. Vacuum filtration steps should be carried out at –17 to –23 inHg for optimum performance. Important: If using the kit for the first time, please read Section 5, "Before Using the Aurum™ Total RNA Fatty and Fibrous Tissue Kit," and Section 6, "Vaccum Manifold Setup and Use With the Column Adaptor Plate" before proceeding.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 15 Warning: Processing larger amounts of starting material may lead to column clogging and reduced RNA purity. It is crucial that the appropriate amount of starting material be used. For samples that are known to be rich in RNA, it is highly recommended that less than the maximum amount of starting material be used so that the binding capacity of the column is not exceeded.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 16 Cells Grown in a Monolayer Cells grown in a monolayer should be lysed with PureZOL directly in the culture dish. Aspirate the culture medium and immediately add 1 ml of PureZOL to a 10 cm2 dish. Pass the lysate through a pipet several times. The amount of PureZOL added is dependent on the area of culture dish (1 ml per 10 cm2) and not on cell number. Insufficient volumes of PureZOL may result in DNA contamination. Proceed to step 3.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 17 4. Add 0.2 ml of chloroform to the lysate, then cover and shake vigorously for 15 sec. Do not vortex! 5. Incubate for 5 min at room temperature while periodically mixing the sample. 6. Centrifuge at 12,000 x g for 15 min at 4°C. Following centrifugation, the mixture will separate into three phases: an upper, colorless aqueous phase, a white interphase, and a lower, red organic phase.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 18 10. Pipet 700 µl of the RNA sample into the RNA binding mini column. Turn the vacuum on and adjust to –17 to –23 inHg by closing the vacuum regulator. Continue to apply vacuum until all of the RNA sample passes through the column. Open the vacuum regulator until the gauge indicates 0 inHg. 11. Repeat step 11 for the remainder of the sample.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 19 18. Pipet 40 µl (or 30 µl)† of the elution solution onto the center of the membrane at the bottom of the RNA binding column. † Note: When isolating total RNA from small amounts of starting material (<10 mg of tissue or 500,000 cells), perform a single elution with 30 µl of the elution solution. Do not perform step 21. 19.Incubate 1 min for complete soaking and saturation of the membrane. 20. Centrifuge for 2 min at >12,000 x g to elute the total RNA.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 20 Section 8 Spin Protocol Important: If using the kit for the first time, please read Section 6, "Before Using the Aurum™ Total RNA Fatty and Fibrous Tissue Kit," before proceeding. Centrifugation steps can be performed on any commercially available microcentrifuge that can accommodate 1.5 and 2.0 ml microcentrifuge tubes and can spin at >12,000 x g. It is recommended that sterile, disposable polypropylene tubes be used throughout the procedure.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 21 up to 100 mg of freshly dissected tissue into a 2.0 ml microcentrifuge tube and add 1 ml of PureZOL. Disrupt the sample for 30–60 sec using a rotor-stator homogenizer or a bead mill homogenizer (refer to manufacturer instructions for more details). Although not as effective, passing the tissue sample through an 18-gauge needle and syringe can be used for sample disruption if a homogenizer is not available.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 22 Note: Do not wash cells prior to the addition of PureZOL as this could increase the possibility of mRNA degradation. 3. Once the sample has been disrupted in PureZOL, incubate the lysate at room temperature for 5 min to allow the complete dissociation of nucleoprotein complexes. Note: Following the disruption step, the sample can be stored at –70°C for at least one month. To process frozen lysates, samples should be thawed at room temperature.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 23 b. For each column to be processed, mix 5 µl of reconstituted DNase I with 75 µl of DNase dilution solution in a 1.5 ml microcentrifuge tube. Scale up proportionally if processing more than one column. Once diluted with DNase dilution solution, do not refreeze for later use. 7. Without disturbing the interphase, immediately transfer aqueous phase to a 2.0 ml microcentrifuge tube.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 24 14.Add 700 µl of high stringency wash solution to the RNA binding column. Centrifuge for 30 sec at >12,000 x g. Discard the high stringency wash solution from the wash tube and place the column back into the same wash tube. 15.Add 700 µl of low stringency wash solution (already supplemented with ethanol) to the RNA binding column. Centrifuge for 1 min at >12,000 x g.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 25 Section 9 Troubleshooting Guide Problems that may be encountered during RNA purification: Problem Possible Cause Recommended Solution Incomplete separation of phases after centrifugation Lysate was not mixed properly after adding chloroform (see step 4 in protocol) Once chloroform is added, mix tubes vigorously by shaking for 15 sec.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 26 Problem Possible Cause Recommended Solution RNA binding mini column is clogging (continued) Starting material is high in fat, proteins, polysaccharides, or extracellular material, causing RNA eluate impurities After the sample disruption step, centrifuge the lysate at 12,000 x g for 10 min at 4°C to pellet any debris present. Transfer the supernatant into a new 2.0 ml microcentrifuge tube, leaving behind the pellet.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 27 Problem Possible Cause Recommended Solution Low RNA yield (continued) Low amount of starting material Do not use less than the recommended minimum starting amount (see Section 5).
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 28 Problem Possible Cause Recommended Solution Genomic DNA contamination On-column DNase I digest was not performed Perform the on-column DNase I digest (see step 14 in protocol) Incomplete DNase I digest Increase the digest time for starting materials that are known to contain a high level of genomic DNA DNase I is inactive Reconstitute the lyophilized DNase I with 10 mM Tris, pH 7.5.
10001298B:4110133A.qxd 1/16/2009 2:40 PM Page 29 Problem Possible Cause Recommended Solution RNA is degraded (continued) Frozen tissue samples were allowed to thaw or sit at room temperature Add PureZOL directly to frozen samples before they thaw. Do not let starting materials sit at room temperature Cells grown in either monolayer or suspension were washed prior to lysis with PureZOL Cells grown in monolayer: aspirate the growth medium and then add PureZOL directly to the plate.
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10001298B:4110133A.qxd 1/16/2009 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
