Complex Plant Total RNA (plus) Small Amount Extraction Kit

1. Components of the reagent kit

2. Storage

This reagent kit can be stored at room temperature (15-25℃) in a dry environment and is stable for 12 months.

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 reagent kit provides a fast and effective purification of plant total RNA containing polysaccharides, lipids, polyphenols, and other components. It is suitable for most complex plant tissues. In general, lipid-rich plant tissues contain a high content of lipid compounds, which significantly affect RNA extraction efficiency. This kit utilizes exclusive inhibitor removal columns to effectively eliminate lipids from plant samples, as well as to remove DNA contamination. If the experiment is sensitive to DNA, it is recommended to use intron-spanning primers for downstream experiments.

The RNA rapid purification reagent kit can extract plant total RNA (including nuclear RNA and cytoplasmic RNA) within 1 hour. The extracted RNA can be directly used for RT-PCR, Northern blotting, etc. The entire purification process does not require toxic reagents such as chloroform, making the RNA purification reagent kit suitable for various other samples.

5. Experimental Principles and Procedures

6. Extraction Process

Precautions before starting the experiment:

A. Wash Buffer 1: Prior to use, add the specified amount of absolute ethanol as indicated on the reagent bottle label. Check the label to confirm the addition of absolute ethanol.

B. Elution Buffer is a 0.1x TE solution with a minimal amount of EDTA. If EDTA may affect subsequent experiments, it is recommended to replace the elution buffer with sterile deionized water.

C. RNA Extraction Buffer I contains a small amount of phenol, which may precipitate. Before use, mix thoroughly by warming in a water bath; after use, store away from light.

1. Sample Processing:

A. Material Collection and Storage:

If freshly collected materials cannot be immediately used, place them in liquid nitrogen and store them at -80℃.

B. Whenever possible, collect fresh materials as they contain fewer polysaccharides and polyphenols.

2. Grind approximately 100 mg of fresh samples or not more than 20 mg of dry material with liquid nitrogen.

3. Add 600μl of RNA Extraction Buffer I, ensuring there are no tissue clumps in the ground sample. Tissue clumps are difficult to lyse and can reduce RNA yield.

4. Vortex for 30 s.

5. Transfer the lysate to aninhibitor removal purification column, centrifuge at 12,000 rpm for 5 min.

(Note: Lipid-rich plant materials may contain many lipid compounds at this step, which can affect RNA extraction. Remove these substances during this step. If buffer residue remains in the inhibitor removal purification column during centrifugation, extend centrifugation time appropriately.)

6. Transfer the obtained supernatant to a DNA removal column, centrifuge at 12,000 rpm for 30s, and collect the filtrate (Note: RNA is present in the filtrate).
7. Add 250μl of absolute ethanol, mix by pipetting. If there is a small amount of precipitation, it does not affect subsequent experiments. Transfer the liquid to an RNA purification column, centrifuge at 12,000 rpm for 30s, discard the flow-through.
8. Add 700μl of inhibitor removal buffer, centrifuge at 12,000 rpm for 30s, discard the flow-through.
9. Add 700μl of washbuffer 1 to the RNA purification column, centrifuge at 12,000 rpm for 30s, discard the flow-through.
10. Add 500μl of wash buffer 1 to the RNA purification column, centrifuge at 12,000 rpm for 3 min, discard the flow-through.
11. Place the RNA purification column into a new 1.5ml centrifuge tube, air-dry the membrane at room temperature for 2 minutes.

(Note: Confirm that absolute ethanol has been added to wash buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so drying the membrane is crucial. After centrifugation, ensure no ethanol is present before elution, then discard the waste and collection tube. After washing with wash buffer 1, the membrane on the RNA purification column should have only a slight color. After centrifugation, carefully remove the RNA purification column, ensuring it does not touch the collection tube to avoid ethanol interference.)

12. Aerially pipette 50-100μl of elution buffer onto the membrane, centrifuge at 12,000 rpm for 1 min, and collect the RNA solution.

(Note: Eluting RNA with 50 μl of elution buffer can increase RNA concentration but reduces total RNA yield.)