Animal Tissue Total RNA Extraction Kit
40-minute rapid extraction, 0 phenol, 0 chloroform
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Instruction manual- Product Description
- Composition
- Precautions
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Transportation and Storage Conditions: Proteinase K is shipped in an ice pack and stored at −20°C; other reagents and spin columns are shipped at room temperature and stored at room temperature (15–25°C). It is recommended to aliquot the elution buffer into small portions before first use and store it at room temperature (15–25°C).
Product Overview:
This kit is designed for the rapid extraction of high‑quality total RNA longer than 200 nt from approximately 20 mg of animal tissue samples. It employs a proprietary lysis buffer that efficiently disrupts biological samples and inactivates RNases, ensuring the integrity of the extracted RNA. The kit offers simple, fast operation with stable and reliable performance; it does not use toxic reagents such as phenol or chloroform, nor does it require time‑consuming alcohol precipitation. The entire extraction process takes about 40 minutes, yielding RNA with high yield and purity, making it suitable for applications including RT‑PCR, RT‑qPCR, Northern blotting, gene expression microarray analysis, and high‑throughput sequencing.
Instructions for Reference Dosage in Animal Tissues
Organization Type Liver, pancreas, intestines, stomach Heart, Spleen, Kidney, Lung, muscle Fat Reference dosage (mg) 10 20-30 20-50 50 The sample size is critical to the extraction performance; select an appropriate sample size whenever possible.
Preparations
1. Before first use, add 40 mL of anhydrous ethanol to Buffer CB and shake thoroughly.
2. Before first use, add 45 mL of anhydrous ethanol to Buffer WB and mix thoroughly.
3. If the tissue sample requires Proteinase K for extraction, preheat a 55°C water bath before starting the experiment.
Operating Steps
Lysis of complex samples and column-loading steps
1. Weigh approximately 20 mg of tissue into a 1.5 ml centrifuge tube, add 350 μl of lysis buffer, and first use scissors to quickly mince the tissue into small pieces. Then homogenize using an electric homogenizer, a glass homogenizer, or a tissue disruptor. Note that the sample should be ground rapidly to ensure thorough and prompt contact between the tissue and the lysis buffer, thereby inhibiting RNase activity.
2. Add 350 μl of Lysis Buffer diluent to the homogenized sample, followed by 20 μl of Proteinase K, and vortex to mix thoroughly.
3. Incubate in a 55°C water bath for 10 minutes.
4. Centrifuge at 14,000 × g for 5 minutes, transfer the supernatant to a new centrifuge tube, and carefully avoid aspirating the pellet.
5. Add 0.5 volume of anhydrous ethanol to the supernatant, and pipette up and down 10 times to mix thoroughly (if precipitation occurs, this is normal; use a pipette to vigorously pipette up and down to resuspend the precipitate).
6. Transfer the mixture to the RNA purification column (with collection tube), centrifuge at 12,000 × g at room temperature for 1 minute, and discard the waste liquid. If the volume of the mixture is large, you may transfer it in two portions.
Simple sample lysis and column-loading steps
1. Weigh approximately 20 mg of tissue into a 1.5 ml centrifuge tube, add 350 μl of lysis buffer, and first use scissors to quickly mince the tissue into small pieces. Then homogenize using an electric homogenizer, a glass homogenizer, or a tissue disruptor. Note that the sample should be processed rapidly to ensure thorough contact between the tissue and the lysis buffer, thereby inhibiting RNase activity.
2. Centrifuge at 14,000 × g for 5 minutes, transfer the supernatant to a new centrifuge tube, and carefully avoid aspirating the pellet.
3. Add an equal volume of 70% ethanol—prepared by diluting with Lysis Buffer—to the supernatant, and pipette up and down 10 times to mix thoroughly (if precipitation occurs, this is normal; use a pipette to vigorously pipette up and down to resuspend the precipitate).
4. Transfer the mixture to the RNA purification column (with collection tube), centrifuge at 12,000 × g at room temperature for 1 minute, and discard the waste liquid. If the volume of the mixture is large, you may transfer it in two portions.
Note: Complex samples refer to tissue specimens that are difficult to lyse or have low RNA content, making extraction challenging. For samples of unknown extractability, the initial lysis and column‑binding steps should be performed as if they were complex samples.
Simple samples: easily lysable tissue samples with high RNA content that are straightforward to extract. For tissues such as liver, intestine, and stomach, which can be readily homogenized after the addition of lysis buffer and yield a high RNA recovery (greater than 15 μg per 20 mg), a simplified protocol may be employed for subsequent extractions by following the lysis and column‑binding steps used for simple samples.
The following column‑washing and RNA‑elution steps are performed identically for both complex and simple samples.
Column Cleaning Procedure
1. Add 350 μl of Buffer CB to the RNA purification column (with collection tube), centrifuge at room temperature at 12,000×g for 1 minute, and discard the waste liquid.
2. Add 350 μl of Buffer CB to the RNA purification column (with collection tube), centrifuge at room temperature at 12,000×g for 1 minute, and discard the waste liquid.
3. Add 500 μl of Buffer WB to the RNA purification column (including the collection tube), centrifuge at room temperature at 12,000×g for 1 minute, and discard the waste liquid.
4. Centrifuge at room temperature at 12,000 × g for 2 minutes.
RNA elution step
5. On a clean bench, transfer the RNA purification column into a clean, RNase-free 1.5 mL centrifuge tube and allow it to air-dry with the cap open at maximum fan speed for 2 minutes.
6. Add 20–30 μl of Elution Buffer to the central region of the RNA purification column membrane and let it stand at room temperature for 2 minutes.
7. Centrifuge at room temperature at 12,000 × g for 1 minute; discard the spin column. The product in the 1.5 mL centrifuge tube is RNA, which should be stored at −80°C.
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Product Composition:
Product Number Component (100 times) 400-103A Lysis Buffer 40 ml 400-103B Buffer CB 40 ml 400-103C Buffer WB 15 ml 400-103D Elution buffer 20 ml 400-103E Proteinase K 2 ml 400-103F Lysis Buffer Diluent 40 ml 400-103G RNA Purification Column (with Collection Tube) 100 sets -
Precautions
1. The extraction process is carried out at room temperature. Use the recommended sample volume to prevent insoluble precipitates from clogging the spin column. After opening the kit, perform each step strictly according to the protocol to avoid RNase contamination, and aliquot the Elution Buffer into small portions for storage.
2. Before first use, add the corresponding volume of anhydrous ethanol to Buffer CB and Buffer WB, and shake thoroughly.
3. To ensure complete RNA dissolution, the volume of the elution buffer should ideally be no less than 20 μl. Repeated elution can increase RNA yield. The elution buffer must be applied directly to the membrane at the center of the RNA purification column; do not apply it to the side walls.
4. To ensure extraction quality, it is recommended to use RNase‑free reagents and consumables throughout the entire process, particularly during the RNA elution step.
Keywords
GOONIE
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