Related Product Information
Directions for purifying RNA from animal and plant cells using the PureLink™ RNA Mini Kit are described below.
For detailed instructions, and protocols for isolating RNA from tissue, blood, bacteria, yeast, liquid samples, and on-column DNAse treatment, download the manual from
www.invitrogen.com or by contacting Technical Support.
- 96–100% ethanol
- 2–mercaptoethanol
- 70% ethanol (in RNase-Free Water)
- 1.5 mL RNase-free microcentrifuge tubes
- RNase-free pipet tips
- Homogenizer, RNase-free syringe (1 mL) with 18–21-gauge needle
- or, Rotor-stator homogenizer
- Microcentrifuge capable of centrifuging 12,000 × g
- PBS (for samples with >107 cells)
- 15 mL RNase-free tubes (for samples with >107 cells)
General Guidelines
Follow proper aseptic RNA handling techniques to prevent RNase contamination of reagents and RNA samples.
- Keep freshly harvested samples on ice and quickly proceed to Lysis and Homogenization, or freeze samples immediately after collection in liquid nitrogen or on dry ice and keep at –80°C for later use.
- Do not exceed the RNA binding capacity of the spin cartridge by adding samples containing more than 1 mg of total RNA.
- Both Lysis Buffer and Wash Buffer I contain guanidine isothiocyanate. Do not add bleach or acidic solutions directly to solutions or sample preparation waste containing guanidinium isothiocyanate, as reactive compounds and toxic gases are generated.
- Solutions containing ethanol are considered flammable. Use appropriate precautions when using this chemical.
12183018A,12183020,12183025
Buffer Preparation
- When using Wash Buffer II for the first time, add 16 mL 96–100% ethanol. Mark the label to indicate that ethanol is already added.
- Prepare fresh Lysis Buffer containing 1% 2-mercaptoethanol. Add 10μL 2–mercaptoethanol for every 1 mL Lysis Buffer
Lysis and Homogenization
Cell Number | Lysis Buffer Required for Each Sample |
≤1 × 106 | 0.3 mL (0.6 mL if using a rotor-stator for lysis/homogenization) |
1 × 106–5 × 106 | 0.6 mL |
5 × 106–5 × 107 | 0.6 mL per 5 × 106 cells (e.g., use 1.2 mL for 1 × 107 cells) |
≤5 × 106 Suspension Cells
- Transfer the cells to an RNase-free tube and centrifuge at 2,000 × g for 5 min at 4°C to pellet. Discard the growth medium.
- Add 0.3 or 0.6 mL Lysis Buffer with 2-mercaptoethanol to the sample (see table above for volume).
- Vortex until the cell pellet is dispersed and the cells appear lysed.
- Proceed to Homogenization below.
≤5 × 106 Monolayer Cells
- Remove the growth medium from the cells, then add 0.3 or 0.6 mL. Lysis Buffer with 2-mercaptoethanol (see table above for volume).
- Vortex until the cell pellet is dispersed and the cells appear lysed.
- Proceed to Homogenization below.
5 × 106–5 × 107 Suspension Cells
- Transfer cells to a 15-mL tube and centrifuge at 2,000 × g for 5 min at 4°C. Discard the supernatant.
- Add 0.6 mL Lysis Buffer with 2-mercaptoethanol (see table above for volume).
- Vortex until the cell pellet is dispersed and the cells appear lysed.
- Homogenize at room temperature with a rotor-stator homogenizer (see Homogenization below).
Frozen Cell Pellets
- Transfer cells to a 15-mL tube and add 0.6 mL Lysis Buffer with 2-mercaptoethanol (see table above for volume).
- Vortex until the cell pellet is dispersed and the cells appear lysed.
- Homogenize at room temperature with a rotor-stator homogenizer (see Homogenization below).
Homogenization
- Proceed with one of the following homogenization options at room temperature:
- Transfer the lysate into a clean homogenization tube, and perform manual homogenization. Centrifuge the homogenate at 12,000 × g for 2 minutes.
- Pass the lysate 5–10 times through an 18- to 21-gauge syringe needle.
- Transfer the lysate into a clean tube, and homogenize using a rotor-stator homogenizer at maximum speed for ≥45 s. Centrifuge the homogenate at 2600 × g for 5 minutes, then transfer the supernatant to a clean RNase-free tube.
- Proceed to RNA Purification
Binding, Washing, and Elution of RNA
- Add one volume 70% ethanol to each volume of cell homogenate.
- Vortex to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol.
- Transfer up to 700 μL of the sample (including any remaining precipitate) to the spin cartridge (with the collection tube).
- Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and reinsert the spin cartridge into the same collection tube.
- Repeat Steps 3–4 until the entire sample has been processed.
- Add 700 μL Wash Buffer I to the spin cartridge.
- Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the collection tube. Place the spin cartridge into a new collection tube.
- Add 500 μL Wash Buffer II with ethanol to the spin cartridge.
- Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through.
- Repeat Steps 8–9 once.
- Centrifuge the spin cartridge at 12,000 × g for 1–2 minutes to dry the membrane with bound RNA. Discard the collection tube and insert the spin cartridge into a recovery tube.
- Add 30–100 μL RNase–free water to the center of the spin cartridge.
- Incubate at room temperature for 1 minute.
- Centrifuge the spin cartridge for 2 minutes at ≥12,000 × g at room temperature to elute the RNA from the membrane into the recovery tube. Note: If the expected RNA yield is >100 μg, perform 3 sequential elutions of 100 μL each. Collect the eluates in a single tube.
- 15. Store your purified RNA or proceed to downstream application.
RNA Storage
Store the purified RNA on ice for immediate use. For long–term storage, keep the purified RNA at –80°C. Perform DNase I treatment after purification (refer to the PureLink™ RNA Mini Kit manual) to assure highly pure RNA without genomic DNA contamination. Determine the quality and quantity of your RNA by UV absorbance at 260 nm.
Problem | Cause | Solution |
---|
Low RNA yield | Incomplete lysis and homogenization
| - Use the appropriate method for lysate preparation based on amount of starting material.
- Decrease the amount of starting material used.
- Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in the Lysis Buffer to achieve optimal lysis.
|
| Poor quality of starting material | - The yield and quality of RNA isolated depends on the type and age of the starting material.
- Use fresh sample and process immediately after collection or freeze the sample at –80°C or in liquid nitrogen immediately after harvesting.
|
| Clogged RNA Spin Cartridge | Clear the homogenate and remove particulate or viscous material by centrifugation. Use only the supernatant for subsequent loading onto the spin cartridge. |
| Ethanol not added to Wash Buffer II | Add ethanol to Wash Buffer II before use |
| Incorrect elution conditions | Add RNase–Free Water (30–100 µL) and incubate for 1 min before centrifugation. To recover more RNA, be sure to use up to 3 sequential elutions of 100 µL each (3 × 100 µL) Elution Buffer |
RNA degraded | RNA contaminated with RNase | Use proper aseptic RNA handling techniques. Use RNase-free plasticware, and wear disposable gloves. Remove RNase contamination from work surfaces and non-disposable items with RNase AWAY® Reagent (cat. no. 10328-011). |
Inhibition of downstream enzymatic reactions
| Presence of ethanol in purified RNA | Traces of ethanol from Wash Buffer II can inhibit downstream enzymatic reactions. Discard Wash Buffer II flow through. Place the spin cartridge into the recovery tube and centrifuge at 12,000 × g for 1–2 min to completely dry the cartridge. |
| Presence of salt in purified RNA
| Use Wash Buffers in the correct order. Always wash with Wash Buffer I followed by Wash Buffer II.
|
Low A260/A280 ratio
| Sample was diluted in water | Use 10 mM Tris–HCl (pH 7.5) to dilute sample for OD measurements.
|
MAN0002725 24-May-2010