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3DNA Array Detection
Protocols |
Genisphere Protocol for RNA Isolation from Animal Cells using Qiagen Rneasy RNA Isolation Kit |
The following protocol is directly taken from Qiagen’s protocol and can be found on their web site (www.qiagen.com/literature/rnalit.asp). We have followed the protocol with only one slight modification: the use of a 16 gauge needle for homogenization instead of the recommended 18-20 gauge.
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| Needed Supplies |
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Qiagen Rneasy Midi Kit |
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14.3 M BME (Fisher Cat#03446I-100) |
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96-100% EtOH |
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Sterile, Rnase-free pipet tips (1 ml) and pipet |
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Sterile, Rnase-free cell scrapers |
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Sterile, Rnase-free 15 ml conical tubes |
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Sterile, Rnase-free 1 ml microfuge tubes |
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Clinical Centrifuge capable of 3000-5000 x g (3600 rpm in a Beckman GS-6 Tabletop) |
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Disposable gloves |
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| Cell Culture |
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| Cells were cultured in T75 flasks. Cells were split out onto 100 mm dishes and harvested at 80-90% confluence. Important: The starting material should range from 5 x 106 to 1 x 108 cells. See the Qiagen protocol for help in determining starting material. |
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| Prior to Isolation |
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Check cells. |
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Add BME to RLT Buffer (10 ml BME per 1 ml of RLT). Make up enough for experiment (2 mls per plate). |
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| Harvesting Cells |
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Aspirate culture media. |
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Add 2 ml of RLT buffer, scrape cells from plate and place in a 15 ml conical tube (not supplied). |
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Homogenize cells by passing through a 16 gauge needle attached to a 3 ml syringe (5-10 times). Qiagen recommends 18-20 gauge needle. We used what we had in stock. |
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| Column Purification |
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Apply the sample to a Rneasy midi spin column placed in a 15-ml tube (supplied) and spin for 5 min. at 3000-5000 x g. Discard the flow-through. |
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Add 4 ml of RW1, spin 5 min (3000-5000 x g). Discard flow through. |
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Add 2.5 ml of RPE, spin 2 min (3000-5000 x g). Discard flow through. |
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Add 2.5 ml of RPE, spin 5 min (3000-5000 x g). Discard flow through. |
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| Elution |
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Transfer Rneasy column to a new 15 ml tube (supplied). |
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Add 150 ml of Rnase-free water to column membrane and spin 3 min. (3000-5000 x g). |
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Repeat with a second volume of Rnase-free water. |
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| Quantitation and QC of RNA |
| Measure absorbance at 260 nm and 280 nm in a spectrophotometer. The following is an example of a quantitation calculation: |
| Dilution=10 ul of RNA + 490 ul of distilled water (1:50 dilution) |
| 1 unit A260=40 mg/ml |
| Absorbance at 260 nm=0.45 |
| Calculation: 50 (diltution factor) x 0.45 units x 40 mg/ml/unit=900 mg/ml or 0.9 mg/ml |
| Yields have ranged from 0.20 to 0.60 mg/ml. An absorbance ratio (A260/ A280) above 1.8 indicates adequate RNA purity. For an in depth explanation, see the Qiagen protocol. |
| To ensure the quality of the RNA, we always run the prepared RNA on a gel. We generally run a 1% denaturing agarose gel. Good quality RNA should result in two distinct bands and little smearing on the lower portion of the gel (see sample gel below). |
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| Storage |
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Aliquot RNA into small quantities (ex. 25 mg) for long term storage. |
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Store at –80oC |
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