Stool Total RNA Extraction Kit

1. Componentes del kit de reactivos

2. Almacenamiento

Este kit de reactivos debe almacenarse a temperatura ambiente. (15-25°C) under dry conditions and can be kept for 12 meses. Lysozyme and Proteinase K contain preservatives, allowing for transportation at room temperature. Para almacenamiento a largo plazo, it should be stored at -20°C.

3. Instrucciones para usar el kit de reactivos

3.1 Este kit está destinado a fines de investigación de biología molecular y no debe utilizarse para el diagnóstico o tratamiento de enfermedades..

3.2 Algunos componentes del kit contienen irritantes.; es recomendable tomar las precauciones necesarias (como usar ropa y gafas protectoras).

3.3 El uso de este kit requiere equipo adicional como una centrífuga de alta velocidad., baño de agua (baño de metal), mezclador Vortex, etanol anhidro, nitrógeno líquido, cloroformo, agua desionizada estéril, y tubos EP.

4. Introducción al kit de reactivos

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, transferencia del sur, etc.. Todo el proceso de purificación no requiere reactivos tóxicos como el fenol-cloroformo., making the RNA purification kit suitable for various other sample types.

5. Principios y procedimientos experimentales

6. Proceso de extracción

Precauciones antes de comenzar el experimento.:

A. Antes de usar, agregue la cantidad especificada de etanol anhidro a LavarBuffer 1 como se indica en la etiqueta del frasco de reactivo, and mark with a check on the label to indicate the addition of anhydrous ethanol.

B. El tampón de elución es un 0.1x solución TE containing a minimal amount of EDTA. If EDTA may affect subsequent experiments, it is recommended to substitute Elution Buffer with sterile deionized water.

1. Procesamiento de muestras (Inhibitor Removal):

A: Add approximately 200mg of samples such as feces, add 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 minutos, centrifugar en 12,000 rpm para 2 minutos, 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, agregar 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 minutos, inverting the tube 6-7 times during digestion.

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

3. Centrifugar en 12,000 rpm para 5 minutos, transfer the supernatant to a new centrifuge tube (approximately 500μl liquid transferred).
4. Add an equal volume of anhydrous ethanol, mezclar bien. 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), incubar a temperatura ambiente durante 2 minutos, centrifugar a más 8,000 rpm para 1 minuto, desechar los residuos recogidos, y vuelva a insertar el tubo de recolección en la columna de purificación para el siguiente paso.
6. Place the RNA purification column into a new collection tube, agregar 400μl of Inhibitor Removal Buffer, centrifugar a más 8,000 rpm para 1 minuto, desechar los residuos, and place the nucleic acid purification column back into the tube for the next step.
7. Agregar 500μl of Wash Buffer 1a la columna de purificación de ARN, centrifugar en 14,000 rpm (20,00×g) para 2 minutos, extending the centrifugation time as needed to ensure membrane drying.

(Nota: The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. Después de la centrifugación, ensure no ethanol is present before elution, luego deseche los residuos y el tubo de recolección..)

8. Place the RNA purification column into a new centrifuge tube, agregar 30µl – 50 μl of Elution Bufferto the membrane, incubar a temperatura ambiente durante 5 minutos (15℃-25℃), centrifugar a más 8,000 rpm para 1 minuto.

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

9. Repeat the previous step.

(Nota: 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.)