Small Amount Tissue microRNA Extraction kit

1. Komponen kit reagen

2. Penyimpanan

Kit reagen ini harus disimpan pada suhu kamar (15-25℃) in a dry condition and is stable for 12 bulan.

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

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 jam. The extracted microRNA can be directly used for RT-PCR, Northern blotting, dll.. The entire purification process does not require toxic reagents such as phenol-chloroform, making the microRNA purification kit suitable for various other samples.

5. Prinsip dan Prosedur Eksperimental

6. Proses Ekstraksi

Tindakan pencegahan sebelum memulai percobaan:

A. Sebelum digunakan, 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. Buffer Elusi adalah a 0.1x solusi TE mengandung sedikit EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water instead of Elution Buffer.

1. Pemrosesan Sampel:

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 tabung sentrifugasi, sentrifugasi di 13,000 rpm untuk 1 menit, collect cells, menambahkan 500μl of RNA Extraction Buffer II, vortex and shake well.

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

3. Vortex for 30 sec.

4. Sentrifugasi lisat untuk 10 min at 14,000 rpm (20,000×g).

(Catatan: 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. Setelah sentrifugasi, transfer the supernatant to a new pemurnian DNA column.)

5. Add the obtained supernatant to the DNA cleanup column (sekitar 650-700μl setiap kali), centrifuge at over 8,000 rpm untuk 1 menit, collect the filtrate (microRNA is in the filtrate at this point).
6. Estimate the volume of the filtrate accurately, menambahkan 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, sentrifugasi di 13,000 rpm untuk 1 menit.
7. Discard the waste and place the RNA purification column in a collection tube for the next step.
8. Menambahkan 600μl of Inhibitor Removal Buffer, centrifuge at over 8,000 rpm untuk 1 menit, membuang limbahnya, and place the RNA purification column back into the collection tube for the next step.
9. Menambahkan 700μl Buffer Cuci 1 to the RNA purification column, sentrifugasi di 14,000 rpm (20,000×g) untuk 2 menit, extend the centrifugation time appropriately to ensure a drier membrane.

(Catatan: Confirm the addition of absolute ethanol to Wash Buffer 1. The presence of ethanol has a severe impact on subsequent experiments. Karena itu, membrane dryness is crucial. Setelah sentrifugasi, ensure the absence of ethanol before elution, membuang limbahnya, and collect the tube.

After washing with Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. Setelah sentrifugasi, 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, menambahkan 100μl Buffer Elusi to the membrane, incubate at room temperature for 5 menit (15℃ to 25℃), centrifuge at over 8,000 rpm untuk 1 menit.

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

11. Repeat the previous step.

Catatan: 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.