UltraPure Total RNA Extraction Kit Spin Column Method For Life Science Research

Product Introduction

The UltraPure Total RNA Rapid Extraction Kit is designed for total RNA extraction from a wide variety of animal and plant tissues as well as cells. The Lysis Buffer RL efficiently lyses tissues and cells while inactivating RNases. In combination with the specific spin columns, it extracts high-purity, high-quality total RNA, free from contaminants such as protein, genomic DNA, and cellular metabolites.

The extracted total RNA exhibits good integrity and is free from protein and DNA contamination. It is suitable for various routine molecular biology experiments, including RT-PCR, Real-Time PCR, Northern blot, Dot blot, poly A selection, in vitro translation, cDNA library construction, and more.

Product Specifications

1. The silica membrane in all spin columns is made of high-quality, specially formulated material, ensuring minimal variation in binding capacity between columns.

2. Combining the stability and high purity of guanidinium thiocyanate/phenol single-step reagents with the convenience of spin columns. Eliminates the need for isopropanol precipitation and ethanol washing steps. RNA is eluted directly from the column, avoiding issues with difficult resuspension due to overdrying.

3. Unique formulation of Lysis Buffer RL effectively eliminates genomic DNA contamination.

4. Multiple column wash steps remove contaminating proteins and cellular metabolites, resulting in high-purity RNA.

5. Effectively reduces the content of 5S RNA in the total RNA, improving purity.

Operation Steps

Pre-experiment Preparation：

1. Use sterile, RNase-free microcentrifuge tubes, pipette tips, and other disposable plastic consumables whenever possible. If using glassware or non-disposable tools, treat them with DEPC or use an RNase decontamination product beforehand.

(1) Soak the tools/containers in a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution, protected from light, for 12 hours.

(2) Autoclave at 121°C for 30 minutes to remove residual DEPC.。

2. Wear a lab coat, disposable latex gloves, and a disposable mask during reagent preparation and experimental procedures to avoid introducing RNase contamination.

Sample Pre-treatment：

1. Tissue Homogenization：

A. Plant Tissue: Grind fresh plant tissue thoroughly in liquid nitrogen, or mince the tissue and grind it directly in Lysis Buffer RL. Add 1 mL of Buffer RL per 50-100 mg of tissue and mix. Note: Sample volume should generally not exceed 10% of the Buffer RL volume.

B. Animal Tissue: Mince fresh or -70°C frozen animal tissue as finely as possible. Add 1 mL of Lysis Buffer RL per 30-100 mg of tissue and homogenize using a homogenizer. Alternatively, grind in liquid nitrogen, then add 1 mL of Buffer RL and mix. Note: Sample volume should generally not exceed 10% of the Buffer RL volume.

C. Monolayer Cultured Cells (Adherent Cells): After removing the culture medium as completely as possible, directly add 1 mL of Lysis Buffer RL to cover the cells in a 3.5 cm culture dish, and lyse by pipetting repeatedly. The amount of Buffer RL required is determined by the culture area, not cell count (add 1 mL per 10 cm²). Insufficient Buffer RL may lead to DNA contamination in the extracted RNA.

Note: Adherent cells may not detach completely from the flask/dish. This does not indicate incomplete lysis, as cell membranes are likely fully ruptured and RNA released. Proceed with the protocol.

D. Suspended Cells: Pellet cells by centrifugation. Lyse the pellet in Lysis Buffer RL by repeated pipetting. Add 1 mL of Lysis Buffer RL per 5~10 x 10⁶ animal, plant, or yeast cells, or per 1 x 10⁷ bacterial cells. Avoid washing cells before adding Buffer RL, as this may increase mRNA degradation. A homogenizer may be required to disrupt certain yeast and bacterial cells.

E. For bacterial RNA extraction, the Bacterial RNA Extraction Kit (Spin Column) (Cat. No. YBR3220) is recommended. For blood RNA extraction, the TRI Whole Blood (Liquid Sample) Total RNA Extraction Kit (Cat. No. YBR3208) is recommended.

Extraction Steps：

1. Vortex the homogenate vigorously and incubate at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes.

2. Optional Step: Centrifuge at 12,000 rpm for 10 minutes at 4°C and transfer the supernatant. This step can remove debris if the sample contains high amounts of protein, fat, polysaccharides, or materials like muscle or plant tubers. The pellet contains cell walls, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. For fatty tissue samples, remove the top lipid layer and use the clear homogenate for the next step.

3. Add 0.2 mL of chloroform per 1 mL of Lysis Buffer RL used. Cap the tube tightly, vortex vigorously for 15 seconds, and incubate at room temperature for 3 minutes.

4. Centrifuge at 12,000 rpm for 10-15 minutes at 4°C in a high-speed refrigerated centrifuge. After centrifugation, the mixture separates into three phases: a lower red organic phase, an interphase, and an upper colorless aqueous phase. RNA is contained in the upper aqueous phase, which has a volume approximately 50-60% of the original Buffer RL volume.

5. Carefully transfer the aqueous phase to a new RNase-free microcentrifuge tube (avoid touching the interphase). Record the volume of the aqueous phase. Add 0.5 volume of anhydrous ethanol (relative to the aqueous phase volume) and mix immediately by pipetting.

6. Transfer the mixture (in aliquots of ≤700 µL if necessary) to the SpinRA column. Centrifuge at 12,000 rpm for 45 seconds. Discard the flow-through and place the column back into the collection tube.

7. Add 500 µL of Deproteinization Buffer RE. Centrifuge at 12,000 rpm for 45 seconds at room temperature. Discard the flow-through.

8. Add 500 µL of Wash Buffer RW (ensure ethanol has been added beforehand). Centrifuge at 12,000 rpm for 45 seconds. Discard the flow-through.

9. Repeat Step 9 once.4

10. Place the SpinRA column back into the empty collection tube. Centrifuge at 13,000 rpm for 2 minutes at room temperature to dry the membrane completely. Removing residual ethanol is crucial as it can inhibit downstream reactions.

11. Transfer the SpinRA column to a new RNase-free microcentrifuge tube (avoid contact between the column bottom and any flow-through). Apply 50-80 µL of RNase-free H2O directly to the center of the membrane. Let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute to elute the RNA. Store the eluted RNA at -80°C to prevent degradation.

Larger elution volumes yield higher elution efficiency. For higher RNA concentration, the elution volume can be reduced, but it is recommended not to use less than 30 µL, as very small volumes reduce elution efficiency and yield.