Small Amount Tissue microRNA Extraction kit

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25℃) in a dry condition 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 microRNA purification kit provides a fast and efficient method for purifying microRNA from various tissues, suitable for most species. Tissues exceeding 100 mg can be processed using this RNA purification kit. The kit employs special DNA cleanup column technology to remove genomic DNA and large RNA fragments during the experimental process. In general, additional digestion on DNA columns is not required, preventing microRNA degradation.

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

5. Experimental Principles and Procedures

6. Extraction Process

Precautions before starting the experiment:

A. Before use, add the specified amount of anhydrous ethanol to Wash Buffer 1 according to the label on the reagent bottle, and mark a check on the label to indicate the addition of anhydrous ethanol.

B. Elution Buffer is a 0.1x TE solution containing a minimal amount of EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water instead of Elution Buffer.

1. Sample Processing:

A. Plant or Animal Tissues: Collect fresh tissues whenever possible. For plant and animal tissues, grind with liquid nitrogen, then quickly add 500μl of RNA Extraction Buffer II. Invert and mix thoroughly, avoiding tissue clumps in the lysate to prevent RNA degradation.

B. Cell Tissues: Collect fresh growing cells in a 1.5 ml centrifuge tube, centrifuge at 13,000 rpm for 1 min, collect cells, add 500μl of RNA Extraction Buffer II, vortex and shake well.

2. Incubate at56℃ for 1-3 min. If the sample has a high polysaccharide content, it is advisable to skip this step.

3. Vortex for 30 sec.

4. Centrifuge the lysate for 10 min at 14,000 rpm (20,000×g).

(Note: Polysaccharides from plant materials may result in sticky substances at this step, which can cut DNA in subsequent steps. Remove these substances during this step. After centrifugation, transfer the supernatant to a new DNA purification column.)

5. Add the obtained supernatant to the DNA cleanup column (approximately 650-700μl each time), centrifuge at over 8,000 rpm for 1 min, collect the filtrate (microRNA is in the filtrate at this point).
6. Estimate the volume of the filtrate accurately, add 0.5 times the volume of absolute ethanol. If there is a small amount of precipitation, it does not affect subsequent experiments. Add the liquid to the RNA purification column, centrifuge at 13,000 rpm for 1 min.
7. Discard the waste and place the RNA purification column in a collection tube for the next step.
8. Add 600μl of Inhibitor Removal Buffer, centrifuge at over 8,000 rpm for 1 min, discard the waste, and place the RNA purification column back into the collection tube for the next step.
9. Add 700μl of Wash Buffer 1 to the RNA purification column, centrifuge at 14,000 rpm (20,000×g) for 2 min, extend the centrifugation time appropriately to ensure a drier membrane.

(Note: Confirm the addition of absolute ethanol to Wash Buffer 1. The presence of ethanol has a severe impact on subsequent experiments. Therefore, membrane dryness is crucial. After centrifugation, ensure the absence of ethanol before elution, discard the waste, and collect the tube.

After washing with Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. After centrifugation, carefully remove the RNA purification column, ensuring it does not touch the collection tube to avoid ethanol interference.)

10. Place the RNA purification column into a new centrifuge tube, add 100μl of Elution Buffer to the membrane, incubate at room temperature for 5 min (15℃ to 25℃), centrifuge at over 8,000 rpm for 1 min.

(Note: Eluting RNA with 50μl of Elution Buffer can increase RNA concentration but decrease total RNA yield.)

11. Repeat the previous step.

Note: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.