Super-Pure RNA Extraction Kit (TRIzol)

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25℃) in a dry environment and is stable for 12 months. DNase I contains a preservative, allowing transportation at room temperature, but for long-term storage, it should be kept at -20℃.

3. Instructions for Using the Reagent Kit

3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.

3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).

3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, liquid nitrogen, chloroform, sterile deionized water, and EP tubes.

4. Introduction to the Reagent Kit

This RNA purification kit utilizes the traditional TRIzol combined with column membrane method for the rapid purification of plant, animal, tissue, cell, microbial RNA, and fungal hyphae RNA. It is suitable for most species. This RNA purification kit can be applied to plant tissues exceeding 100 mg. The RNA extracted with this kit has extremely low DNA content. If sensitivity to DNA in experiments is a concern, it is recommended to use DNase I digestion on the column.

The RNA Fast Purification Kit can extract total RNA from samples (including nuclear RNA and cytoplasmic RNA) within 1 hour. The extracted RNA can be directly used for RT-PCR, Northern blotting, and other applications.

5. Experimental Principles and Procedures

6. Extraction Process

Precautions before starting the experiment:

A. Before use, add the specified amount of absolute ethanol to WashBuffer 1 according to the label on the reagent bottle, and check the box on the label to indicate that absolute ethanol has been added.

B. Elution Buffer is a 0.1x TE solution containing minimal EDTA. If EDTA affects subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Sample Processing:

A. Tissue: If fresh materials cannot be used immediately, place them in liquid nitrogen and store them at -80°C. Dried materials can be stored at room temperature. Grind 30~80 mg of tissue in liquid nitrogen and add 1 ml Trizol buffer for homogenization.

B. Monolayer Cultured Cells: Aspirate the culture medium and add 1 ml Trizol buffer for homogenization.

C. Cell Suspension: Centrifuge to collect cells and add 1 ml Trizol buffer for homogenization.

2. After adding Trizol buffer to the sample, mix thoroughly by pipetting, allow complete sample lysis, and incubate at room temperature for 5 minutes.

3. Add chloroform in a ratio of 200 μl per 1 ml of Trizol buffer, vigorously shake for 30 seconds, and incubate at room temperature for 2 minutes.

4. Centrifuge the lysate at 4°C, 12,000 rpm for 10 minutes. RNA will be present in the upper aqueous phase.

5. Carefully transfer the supernatant to a new centrifuge tube, avoiding the collection of the middle and bottom layers. (Note: Approximately 400 μl of liquid can be transferred, which may be less for some species.)

6. Add an equal volume of pre-chilled absolute ethanol, mix quickly. (For example, add 400 μl of lysate, add 400 μl of absolute ethanol. If the lysate volume is less than 400 μl, reduce the amount of absolute ethanol proportionally. A slight precipitate may form after adding ethanol, but it does not affect subsequent experiments.)

7. Transfer the obtained liquid to an RNA purification column (approximately 650-700 μl per time), centrifuge at more than 8,000 rpm for 1 minute, discard the collected waste, and reinsert the collection tube into the purification column for the next step.

8. Repeat step 7, adding the remaining liquid to the RNA purification column, centrifuge at more than 8,000 rpm for 1 minute, discard the waste and the collection tube.

9. Place the RNA purification column in a new collection tube, add 300 μl of Inhibitor Removal Buffer, centrifuge at more than 8,000 rpm for 1 minute, discard the waste, and reinsert the RNA purification column into the tube for the next step.

10. Add 80 μl of DNase Iworking solution to the RNA purification column, incubate at 20°C to 30°C for 15 minutes. (Prepare DNase I working solution: 62 μl RNase-Free Water, 8 μl 10× Reaction Buffer, and 10 μl DNase I, make up to 80 μl DNase I working solution.)

11. Add 300 μl of Inhibitor Removal Bufferto the RNA purification column, centrifuge at more than 8,000 rpm for 1 minute, discard the waste, and reinsert the RNA purification column into the tube for the next step.

12. Add 700 μl of Wash Buffer 1to the RNA purification column, centrifuge at 14,000 rpm (20,000×g) for 2 minutes, extend the centrifugation time if needed for a drier membrane. (Note: Confirm the addition of ethanol to Wash Buffer 1; ethanol presence significantly affects subsequent experiments. Ensure the membrane is dry after centrifugation before elution. Discard the waste and the collection tube. After using Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. Carefully remove the RNA purification column after centrifugation, ensuring it does not touch the collection tube to avoid ethanol contamination.)

13. Place the RNA purification column in a new centrifuge tube, drip 100 μl of Elution Bufferonto the membrane, incubate at room temperature for 5 minutes (15°C to 25°C), and centrifuge at more than 8,000 rpm for 1 minute. (Note: Eluting RNA with 50 μl of Elution Buffer can increase RNA concentration but decrease total RNA yield.)

14. Repeat the previous step. (Note: A new centrifuge tube can be used to collect the RNA eluted the second time or continue using the original collection tube to collect RNA.)