Stool Total RNA Extraction Kit

1. Komponen kit reagen

2. Penyimpanan

Kit reagen ini harus disimpan pada suhu kamar (15-25°C) under dry conditions and can be kept for 12 bulan. Lysozyme and Proteinase K contain preservatives, allowing for transportation at room temperature. For long-term storage, it should be stored at -20°C.

3. Petunjuk Penggunaan Kit Reagen

3.1 Kit ini ditujukan untuk tujuan penelitian biologi molekuler dan tidak boleh digunakan untuk diagnosis atau pengobatan penyakit.

3.2 Beberapa komponen dalam kit mengandung bahan pengiritasi; disarankan untuk mengambil tindakan pencegahan yang diperlukan (seperti memakai pakaian pelindung dan kacamata).

3.3 Penggunaan kit ini memerlukan peralatan tambahan seperti centrifuge berkecepatan tinggi, mandi air (mandi logam), pencampur pusaran, etanol anhidrat, nitrogen cair, khloroform, air deionisasi steril, dan tabung EP.

4. Pengantar Kit Reagen

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, Blot Selatan, dll.. 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. Prinsip dan Prosedur Eksperimental

6. Proses Ekstraksi

Tindakan pencegahan sebelum memulai percobaan:

A. Prior to use, add the specified amount of anhydrous ethanol to MencuciPenyangga 1 as indicated on the reagent bottle label, and mark with a check on the label to indicate the addition of anhydrous ethanol.

B. Buffer Elusi adalah a 0.1x solusi TE mengandung sedikit EDTA. If EDTA may affect subsequent experiments, it is recommended to substitute Elution Buffer with sterile deionized water.

1. Pemrosesan Sampel (Inhibitor Removal):

A: Add approximately 200mg of samples such as feces, add 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 menit, sentrifugasi di 12,000 rpm untuk 2 menit, dan buang supernatannya.

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, menambahkan 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 menit, inverting the tube 6-7 times during digestion.

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

3. Sentrifugasi di 12,000 rpm untuk 5 menit, pindahkan supernatan ke tabung centrifuge baru (approximately 500μl liquid transferred).
4. Add an equal volume of anhydrous ethanol, aduk rata. Precipitation may occur, but it does not affect subsequent experiments.
5. Transfer the obtained liquid to an RNA purification column (sekitar 650-700μl setiap kali), incubate at room temperature for 2 menit, centrifuge di atas 8,000 rpm untuk 1 menit, membuang sampah yang dikumpulkan, dan masukkan kembali tabung pengumpul ke dalam kolom pemurnian untuk langkah selanjutnya.
6. Place the RNA purification column into a new collection tube, menambahkan 400μl of Inhibitor Removal Buffer, centrifuge di atas 8,000 rpm untuk 1 menit, membuang limbahnya, and place the nucleic acid purification column back into the tube for the next step.
7. Menambahkan 500μl Buffer Cuci 1to the RNA purification column, sentrifugasi di 14,000 rpm (20,00×g) untuk 2 menit, extending the centrifugation time as needed to ensure membrane drying.

(Catatan: The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. Setelah sentrifugasi, 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, menambahkan 30μl – 50 μl Buffer Elusito the membrane, incubate at room temperature for 5 menit (15℃-25℃), centrifuge di atas 8,000 rpm untuk 1 menit.

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

9. Repeat the previous step.

(Catatan: 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.)