Stool Total RNA Extraction Kit

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25°C) under dry conditions and can be kept for 12 months. Lysozyme and Proteinase K contain preservatives, allowing for transportation at room temperature. For long-term storage, it should be stored at -20°C.

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

The Fecal Total RNA Purification Kit provides a fast and efficient method for purifying total RNA from feces and vomit, widely applicable to the extraction of Gram-positive bacteria, Gram-negative bacteria, and viral RNA in feces and vomit samples.

This kit effectively removes impurities and inhibitors from feces and vomit, reducing inhibition in subsequent downstream experiments such as PCR. The extracted RNA can be directly used for PCR, Southern blotting, etc. The entire purification process does not require toxic reagents such as phenol-chloroform, making the RNA purification kit suitable for various other sample types.

5. Experimental Principles and Procedures

6. Extraction Process

Precautions before starting the experiment:

A. Prior to use, add the specified amount of anhydrous ethanol to WashBuffer 1 as indicated on the reagent bottle label, and mark with 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 may affect subsequent experiments, it is recommended to substitute Elution Buffer with sterile deionized water.

1. Sample Processing (Inhibitor Removal):

A: Add approximately 200mg of samples such as feces, add 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 minutes, centrifuge at 12,000 rpm for 2 minutes, and discard the supernatant.

B: If the extracted sample is a virus sample, you can proceed directly to step 2. If the sample contains a high amount of inhibitors, it is recommended to perform the inhibitor removal step first, although it may reduce the yield of viral nucleic acid.

2. Sample Lysis:

A: For extracting nucleic acids from bacterial samples, add 560μl of Buffer C Plus, 20μl Proteinase K, and 20μl Lysozyme to the centrifuge tube from the previous step. Invert and mix thoroughly, digest at 85℃ for 5 minutes, inverting the tube 6-7 times during digestion.

B: For extracting nucleic acids from virus samples, add 560μl of Buffer C Plus and 20μl Proteinase K. Digest at 85℃ for 5 minutes, inverting the tube 6-7 times during digestion.

3. Centrifuge at 12,000 rpm for 5 minutes, transfer the supernatant to a new centrifuge tube (approximately 500μl liquid transferred).
4. Add an equal volume of anhydrous ethanol, mix thoroughly. Precipitation may occur, but it does not affect subsequent experiments.
5. Transfer the obtained liquid to an RNA purification column (approximately 650-700μl each time), incubate at room temperature for 2 minutes, centrifuge at over 8,000 rpm for 1 minute, discard the collected waste, and reinsert the collection tube into the purification column for the next step.
6. Place the RNA purification column into a new collection tube, add 400μl of Inhibitor Removal Buffer, centrifuge at over 8,000 rpm for 1 minute, discard the waste, and place the nucleic acid purification column back into the tube for the next step.
7. Add 500μl of Wash Buffer 1to the RNA purification column, centrifuge at 14,000 rpm (20,00×g) for 2 minutes, extending the centrifugation time as needed to ensure membrane drying.

(Note: The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. After centrifugation, ensure no ethanol is present before elution, then discard the waste and collection tube.)

8. Place the RNA purification column into a new centrifuge tube, add 30μl – 50 μl of Elution Bufferto the membrane, incubate at room temperature for 5 minutes (15℃-25℃), centrifuge at over 8,000 rpm for 1 minute.

(Note: Using 30μl of Elution Buffer to elute RNA can increase RNA concentration but may reduce total RNA yield.)

9. Repeat the previous step.

(Note: A new centrifuge tube can be used to collect the RNA eluted in the second wash or continue using the original collection tube to collect RNA.)