This product is a dedicated kit for ultra‑micro RNA extraction, suitable for extracting total RNA from ultra‑micro amounts of cells, tissues, insects, plants, fungi, bacteria, and other samples. The typical processing range is 10³–10⁶ cells or <3 mg of tissue. This kit is safe, efficient, rapid, and convenient. It includes a proprietary DNA removal column and RNA lysis/binding reagents, yielding RNA free of DNA contamination. The optimized Trace RNA Enhancer improves RNA extraction quality. The extracted RNA can be directly used in various molecular biology experiments, including Northern blot, dot blot, mRNA purification, in vitro translation, RNase protection assay, RT‑PCR, real‑time RT‑PCR, and cDNA library construction.
Precautions
Operators should wear disposable masks and gloves, change gloves frequently during the experiment, and use RNase‑free plasticware and tips to avoid RNase contamination.
This kit removes most DNA contamination, allowing RNA to be used directly in downstream experiments without DNase I digestion. However, nucleic acid content varies significantly among different samples or different input amounts. If downstream experiments require very high RNA purity, optional DNase I digestion can be performed.
Fresh samples are recommended for RNA extraction. If extraction cannot be performed immediately, samples should be frozen in liquid nitrogen and stored below -70°C. Avoid repeated freeze‑thaw cycles, as this will affect RNA yield and quality.
For micro samples such as nematodes, mites, and pollen, homogenization tube C must be purchased separately.
Protocol
Preparation before use: Add 48 mL of absolute ethanol to 12 mL of Buffer RW2.
1. Homogenization
Add ground plant, animal, fungal, or other samples (bacterial and cell samples do not require grinding) to a 1.5 mL RNase‑Free centrifuge tube pre‑filled with 600 μL Buffer MCL, 10 μL Proteinase K, and 5 μL Trace RNA Enhancer. Immediately vortex vigorously for 30 seconds to lyse thoroughly. Centrifuge at 14,000 rpm (20,000 × g) for 2 minutes.
Note: For micro samples such as nematodes, mites, and pollen, add 600 μL Buffer MCL and 10 μL Proteinase K to homogenization tube C, then add the tissue sample. Grind using a tissue grinder for 30–60 seconds, twice (adjust the machine frequency to avoid breaking the tube). Add 5 μL Trace RNA Enhancer, immediately vortex vigorously for 30 seconds to mix and lyse thoroughly, then centrifuge at 14,000 rpm (18,630 × g) for 2 minutes.
2. Transfer all supernatant to a Genomic DNA Removal Column DE (placed in a collection tube). Centrifuge at 14,000 rpm (18,630 × g) for 30 seconds. Discard the removal column and keep the flow‑through.
3. Add 0.7 volumes of Buffer RB relative to the flow‑through volume (precipitation or flocculent material may appear – this is normal). Mix well, then transfer the mixture to a Spin Column RM (placed in a collection tube). Centrifuge at 14,000 rpm (18,630 × g) for 2 minutes. Discard the flow‑through and return the column to the collection tube.
4. Add 700 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
5. Add 500 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
6. Repeat step 5.
7. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
8. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and let stand at room temperature for 1 minute to completely air‑dry any residual ethanol. Add 10–15 μL RNase‑Free ddH₂O to the center of the membrane without touching it. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. The obtained RNA can be used directly in downstream experiments or stored below -80°C.
Note: The volume of RNase‑Free ddH₂O should not be less than 10 μL; smaller volumes will reduce recovery. To increase RNA yield, reload the first eluate onto the column for a second elution, or preheat the RNase‑Free ddH₂O to 65°C.
RNA Integrity, Concentration, and Purity Assessment
Integrity:
RNA integrity can be assessed by standard agarose gel electrophoresis (conditions: 1–1.2% gel, 0.5× TBE running buffer, 120–150 V for 15–25 minutes). Since rRNA accounts for more than 80% of total RNA, very distinct rRNA bands should be visible under UV after electrophoresis. The types and sizes of rRNA vary among species:
Animals: 28S (4.7–5.0 kb), 18S (1.9 kb), 5S (120 bp)
Plants: 25S (3.7 kb), 18S (1.9 kb), 5S (120 bp)
E. coli: 23S (2.9 kb), 16S (1.5 kb), 5S (120 bp)
Insects/Mollusks: The 18S band is typically brighter than the 28S band.
If the bands are smeared and diffuse, it generally indicates severe RNA degradation. Re‑run the gel or re‑extract the RNA. Otherwise, qPCR may still yield Ct values, but accuracy and reproducibility will be poor.
Concentration:
Determine RNA concentration by measuring absorbance at 260 nm (A₂₆₀) using a spectrophotometer. Use RNase‑Free ddH₂O for blanking. One absorbance unit at 260 nm corresponds to 40 μg/mL RNA.
Purity:
The A₂₆₀/A₂₈₀ ratio is used to assess nucleic acid purity. A ratio between 1.8 and 2.1 indicates high‑purity RNA. The A₂₆₀/A₂₈₀ ratio is dependent on pH and ionic strength; as pH increases, A₂₈₀ decreases while A₂₆₀ remains unchanged, leading to an increased ratio. Because water is typically acidic, it can lower the A₂₆₀/A₂₈₀ ratio. We recommend using a weakly alkaline buffer (e.g., TE, pH 8.0) as the diluent and blank control to obtain accurate, reproducible readings.
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