Ultrapure Total RNA Extraction Kit, IC-5305
Product Introduction
This product is a complete kit for the rapid isolation and purification of high-quality total RNA from a wide variety of biological samples. The kit represents an optimized integration of the classic acidic phenol-guanidinium thiocyanate extraction method with silica membrane-based column purification, providing the high recovery and purity of traditional organic extraction with the speed and convenience of spin column technology. The unique Buffer EX component effectively replaces chloroform in the phase separation step, eliminating the need for toxic halogenated organic solvents and enabling the entire procedure to be performed safely on the laboratory bench without a fume hood. Total RNA purified using this kit is of exceptional quality with minimal genomic DNA contamination and is free of proteins, salts, and other impurities. The purified RNA is suitable for a broad range of downstream applications including RT-PCR, quantitative real-time RT-PCR, Northern blotting, microarray analysis, and RNA sequencing.
Product Features
1. Rapid Protocol: Complete total RNA isolation and purification in 20-40 minutes, significantly faster than traditional organic extraction methods.
2. Chloroform-Free Extraction: Proprietary Buffer EX replaces chloroform in the phase separation step, eliminating the need for toxic halogenated solvents and fume hood operation.
3. Optimized Hybrid Methodology: Combines the high recovery of acidic phenol-guanidinium extraction with the purity and convenience of silica membrane column purification.
4. Broad Sample Compatibility: Validated for total RNA extraction from animal tissues, cultured cells, plant tissues, blood, and other biological samples.
5. High-Quality RNA: Purified RNA is free of genomic DNA, proteins, and salts, suitable for RT-PCR, Northern blot, microarray analysis, and other sensitive downstream applications.
Specifications
Size: 50 extractions
Sample Types: Animal tissues, cultured cells, plant tissues, blood, and other biological samples
Methodology: Acidic phenol-guanidinium thiocyanate extraction with silica membrane column purification
Buffer EX: Chloroform substitute for phase separation
Downstream Applications: RT-PCR, quantitative real-time RT-PCR, Northern blot, microarray analysis, RNA sequencing
Storage and Stability
Storage Conditions: Store kit components at room temperature (15-25°C). After addition of ethanol to Buffer EX, store at 4°C. Protect from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed.
Protocol (For Reference Only)
Important: Work in an RNase-free environment throughout the procedure. Use RNase-free pipette tips, tubes, and gloves. Clean work surfaces with RNase decontamination solution. Pre-chill all reagents and work quickly to minimize RNA degradation.
1. Sample Lysis: For animal tissue, homogenize 10-50 mg of fresh or frozen tissue in 1 mL of Lysis Buffer using a homogenizer or mortar and pestle. For cultured cells, add 1 mL of Lysis Buffer directly to 1×10⁷ cells after removing culture medium and washing with PBS. For plant tissue, grind 50-100 mg of tissue to a fine powder in liquid nitrogen before adding Lysis Buffer. For blood, add 0.25 mL of whole blood to 0.75 mL of Lysis Buffer and mix thoroughly by pipetting.
2. Phase Separation: Add 200 µL of Buffer EX per 1 mL of Lysis Buffer used. Shake vigorously for 15 seconds and incubate at room temperature for 5 minutes. Centrifuge at 12,000 x g for 10 minutes at 4°C. After centrifugation, the mixture separates into an upper aqueous phase containing RNA, an interphase, and a lower organic phase.
3. RNA Binding: Carefully transfer the upper aqueous phase to a clean RNase-free tube without disturbing the interphase. Add an equal volume of 70% ethanol and mix thoroughly by pipetting. Transfer the entire mixture to a spin column placed in a collection tube. Centrifuge at 10,000 x g for 30 seconds at room temperature. Discard the flow-through.
4. Column Washing: Add 700 µL of Wash Buffer 1 to the spin column. Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through. Add 500 µL of Wash Buffer 2 (ensure ethanol has been added) to the spin column. Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through. Repeat the Wash Buffer 2 wash step once.
5. Drying and Elution: Centrifuge the empty spin column at 12,000 x g for 2 minutes to remove residual ethanol. Transfer the spin column to a clean RNase-free 1.5 mL microcentrifuge tube. Add 30-50 µL of RNase-Free Water or Elution Buffer directly to the center of the membrane. Incubate at room temperature for 1-2 minutes. Centrifuge at 10,000 x g for 1 minute to elute the purified RNA. Store the eluted RNA at -80°C or proceed immediately to downstream applications.
Precautions
1. Maintain RNase-free conditions throughout the procedure. Use dedicated RNase-free reagents, consumables, and equipment.
2. Buffer EX must be pre-mixed with ethanol before first use as indicated in the kit instructions. Store at 4°C after ethanol addition.
3. Do not allow the spin column membrane to dry out completely during the procedure; proceed promptly between steps.
4. For samples with high genomic DNA content, an optional on-column DNase I digestion step may be performed between Wash Buffer 1 and Wash Buffer 2 steps.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: How does Buffer EX replace chloroform?
A1: Buffer EX is a proprietary non-toxic phase separation reagent that effectively substitutes for chloroform in the acidic phenol-guanidinium thiocyanate extraction. It enables clean separation of the aqueous RNA-containing phase from the organic phase without requiring halogenated solvents or fume hood operation.
Q2: What is the expected RNA yield and purity?
A2: RNA yield depends on the sample type, quantity, and quality. Typical yields range from 10-50 µg of total RNA from 10 mg of animal tissue or 1×10⁷ cultured cells. The A260/A280 ratio of purified RNA is typically 1.8-2.1, indicating high purity suitable for downstream applications.
Q3: Can this kit be used for small RNA extraction?
A3: Yes. The kit recovers total RNA including small RNA species (<200 nucleotides) such as miRNA, siRNA, and 5S rRNA. For enrichment of small RNAs, a modified protocol with adjusted ethanol concentration may be required.
Q4: How should I store the purified RNA?
A4: Purified RNA should be stored at -80°C in RNase-Free Water or Elution Buffer. For short-term use (up to 1 week), RNA may be stored at -20°C. Avoid repeated freeze-thaw cycles as these promote RNA degradation. For long-term storage, add RNase inhibitor and store at -80°C.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological samples, optimization of homogenization and lysis conditions may be required for specific sample types.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product. Use RNase-free consumables and maintain RNase-free technique.
Ordering Information
Catalog Number: IC-5305
Product Name: Ultrapure Total RNA Extraction Kit
Size: 50 extractions
Price: CNY ¥950.00 / USD $95.00 / EUR �114.00 / JPY ¥17100.00
2023 Version
