Total RNA Isolation Reagent

The Total RNA Isolation Reagent Kit provides an efficient 3-step method to isolate the total RNA from the tissue, cultured animal and bacterial cells, blood, and serum. This unique reagent system ensures the total RNA with a high yield and good quality from samples of unlimited size. If a larger sample is required, the reagent volume can be scaled proportionately, making this reagent not only very user-friendly but also highly versatile. The RNA phenol extraction is not required, and the entire procedure can be completed in 60 minutes. The total RNA is ready for use in RT-PCR, Northern Blotting, cDNA Synthesis and Mapping.

Kit Contents

• Fast procedure delivering high-quality total RNA.
• Ready-to-use RNA for high performance in any downstream application.
• Consistent RNA yield from the starting material with a small amount.
• Provide sufficient reagents and 3 steps to treat the samples.
• Sample: Tissue, cultured animal and bacterial cells, blood, and serum.

Sample Preparation

• Tissue ↘

• Cultured Animal/Bacterial ↘

• Fresh Blood/Frozen Blood ↘

Step 1 Lysis

• Tissue

1. Add 500 μl of the GR Buffer 1 and 8 μl of the ß-mercaptoethanol to the sample in the mortar and grind the sample until it is completely dissolved.
2. Transfer the dissolved sample to a 1.5 ml microcentrifuge tube.

• Cultured Animal and Bacterial Cells/Fresh Blood/Frozen Blood

Add 500 μl of the GR Buffer 1 and 8 μl of the ß-mercaptoethanol to the sample and mix completely

• Serum

1. Transfer 100 μl of the serum to a 1.5 ml microcentrifuge tube.
2. Add 500 μl of the GR Buffer 1 and 8 μl of the ß-mercaptoethanol and mix completely.

Step 2 Phase Separation

1. Add a 1/10 volume of the GR Buffer 2 and 500 μl of the chloroform to the supernatant from the Step 1.
2. Shake vigorously and then centrifuge at 2-8°C at 14-16,000 x g for 10 minutes. Care fully remove the upper phase and transfer it to a new 1.5 ml microcentrifuge tube.
3. Repeat the Phase Separation Step until the interphase becomes clear, then transfer the clear upper phase to a new 1.5 ml microcentrifuge tube.

NOTE: The number of repetitions is dependent on the sample type; e.g. the dense tissue samples may require a higher number of repeats.

Step 3 RNA Precipitation

1. Add 500 μl of the isopropanol to the 1.5 ml microcentrifuge tube containing the clear upper phase from the Step2.
2. Mix the sample by inverting gently and Incubating on the ice for 10 minutes.
3. Centrifuge at 2-8°C at 14-16,000 x g for 15 minutes.
4. Discard the supernatant and wash the pellet with 1 ml of the 70% EtOH.
5. Centrifuge at 2-8°C at 14-16,000 x g for 5 minutes.
6. Completely discard the supernatant and re-suspend the pellets in 50-100 μl of the RNase-free H2O. Incubate for 10 minutes at 60°C to dissolve the pellet.

• Check buffers before use for salt precipitation. Re-dissolve any precipitate by warming up to 37°C.
• During the operation, always wear a lab coat, disposable gloves, and protective equipment.
• Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.