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Preparing Protocol for Measured Samples of 8-OHdG from Specimens |
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Method of Extracting Nucleus DNA from Specimens (for ELISA) |
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| (1) |
Cut the organ specimen (300 mg) into pieces under physiologic saline (2 - 3 ml) cooled with ice and extract the blood. |
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| (2) |
Homogenize in 0.3M glucose solution (2ml) cooled with ice (use physiologic saline if all DNA is to be extracted) and transfer into a 2 ml tube. Rinse twice with 1 ml sucrose solution (use physiologic saline if all DNA is to be extracted) and transfer into a 2 ml tube. |
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| (3) |
Centrifuge (3,000 rpm, for 10 - 20 minutes, at 4 degrees centigrade), remove supernatant, and then rinse the pellet twice with 1 ml physiologic saline. |
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| (4) |
Add proteinase K (Merck: 50μl of 25 mg/ml solution) and SDS (400μl of 1% SDS/1m MEDTA, pH 8.0), cover with argon and incubate at 37 degrees centigrade for 1 - 2 hours (interfusing at intervals). |
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| (5) |
Add 7M Nal (500μl) and isopropyl alcohol (1 ml), cover with argon, and stand at -20 degrees centigrade for at least 10 minutes. |
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| (6) |
Centrifuge (11,000 rpm, for 10 minutes, at 4 degrees centigrade) and rinse the pellet 3 times with cold 70% EtOH (1 ml). |
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| (7) |
Add 0.01xSSC/1mM EDTA (500μl), add RNase solution (2μl), cover with argon, and incubate at 37 degrees centigrade for at least 30 minutes. |
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| (8) |
Add Sevag (600μl) and mix, cover with argon, centrifuge (12,000 rpm, for 10 minutes, at 4 degrees centigrade), and transfer the supernatant by pipette to another tube. |
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| (9) |
Add PEG solution (500μl) and after covering with argon, stand at 4 degrees centigrade overnight. |
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| (10) |
Centrifuge (12,000 rpm, for 15 minutes, at 4 degrees centigrade) and rinse the pellet twice with cold 70% EtOH (1 ml). |
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| (11) |
Add purified water (100μl) and cover with argon, then stand in a refrigerator overnight. |
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| (12) |
Dilute 2 μl of DNA solution with TE 98 μl and measure the 230nm, 260nm, 280nm, and 320nm absorbances with a UV spectrometer to check the concentration and purity of the DNA solution (320nm background absorbance should be subtracted from each wave length. See Example *1). |
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| (13) |
After covering with argon, store at 4 degrees centigrade if hydrolyzed immediately, at -20 degrees centigrade if hydrolyzed within a week, and at -80 degrees centigrade if left longer than a week. |
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How to Hydrolyze DNA |
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Dilute 2 μl of DNA solution with TE 98 μl and measure the 230nm, 260nm, 280nm, and 320nm absorbances with a UV spectrometer to check the concentration and purity of the DNA solution (*1 below). |
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| (1) |
Add purified water to 200 μg DNA solution until the volume reaches 135 μl. |
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| (2) |
Add 200 nM sodium acetate (15 μl) and nuclease P1 (in the case of 6 units:407 units/mg, 15μl of the solution after dissolving in 1ml of purified water) and after covering with argon, incubate at 37 degrees centigrade for 30 - 60 minutes. |
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| (3) |
Add 1M Tris-HCI (18μl; pH 7.4) and alkaline phosphatase (7μl of 2 units : 200 units/0.7 ml) and after covering with argon, incubate at 37 degrees centigrade for 30 - 60 minutes. |
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| (4) |
Centrifuge hydrolysate 14,000 rpm for 10 minutes using Ultrafree-C3LGC to filter (except protein). |
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| (5) |
Analytical curves can be measured if 50μl (theoretically, 200 μg x 50 μl / 187 μl = 53.5 μg DNA) is used in each well as the sample for ELISA. (Adjust the volume as appropriate for each sample if out of the measurable range.) |
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1 To Check the Concentration and Purity of DNA Solution |
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The absorbance 1.0 at 260 nm corresponds to DNA 50 μg/ml. Based on this, calculate DNA concentration. |
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DNA Concentration = Absorbance x 50 (μg/ml) x Dilution
(Example: If the absorbance at 260 nm is 0.55 and the absorbance at 320 nm is 0.05,
The concentration of DNA solution (μg/ml) = (0.55 - 0.05) x 50 x 50 = 1250)
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Estimation of Purity: Ratio, Absorbance at 260 nm / Absorbance at 280 nm = 1.80 -1.85
(Range of High Purity: Ratio, Absorbance at 260 nm / Absorbance at 230 nm = 2.2 - 2.5)
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Check 8-OHdG in Solution |
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8-OHdG; λmax; 245nm, 293nm (MW:283.24) |
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Reagents Used in Tests |
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| All the Water |
Purified water |
| Glucose Solution |
0.3M Sucrose/5mM Tris-HCl pH 7.4, 40μM EDTA |
| 1XSSC |
0.15M NaCl/0.015M Na citrate |
| 0.01XSSC/1mM EDTA |
Calculated 1XSSC and 1M EDTA was diluted with purified water |
| RNase Solution |
(RNase T1=50u,RNase A=100μg)/5μl 50mM Tris- |
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HCl,pH 7.4 |
| Sevag |
Isoamyl alcohol/chloroform = 1/24 |
| TE |
1mM EDTA in 10mM Tris-HCl (pH=8.0) |
| PEG Solution |
PEG8000 (molecular weight 7000 - 8000) 13% (W/V)1.6M Nacl |
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(Provided by Dr. Kaneko at Tokyo Metropolitan Institute of Gerontology) |
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