TRIzol reagent mainly contains phenol and guanidine isothiocyanate. These components rapidly lyse cells while inhibiting the activity of RNases released from the sample. TRIzol disrupts cells and denatures proteins, enabling the separation of nucleic acids from proteins. The pH of the solution determines whether DNA and RNA are partitioned into the aqueous or organic phase. At pH ≥7.0, both DNA and RNA are present in the aqueous phase. When the pH is <7.0, DNA becomes denatured and moves into the interphase or organic phase, while RNA remains in the aqueous phase.
TRIzol reagent is acidic (around pH 5), allowing RNA to remain in the upper aqueous layer during phase separation. After the addition of chloroform and centrifugation, the aqueous phase can be separated and RNA precipitated using isopropanol.
Figure 1: Phase separation during mRNA extraction using TRIzol reagent
TRIzol is widely applicable to many types of samples, but abnormal phenomena may occur during extraction. Below are common issues, possible causes, and suggested solutions:
1. TRIzol appears cloudy when taken out of the refrigerator but becomes clear later.
This is typically normal. TRIzol contains organic components such as phenol and guanidine isothiocyanate, which may become cloudy at low temperatures. If the TRIzol bottle is stored in colder areas of the refrigerator, especially near the back wall where temperatures may drop below 4°C, cloudiness may occur. This does not usually affect performance. To avoid this, store TRIzol near the refrigerator door where temperatures are more stable.
2. Insoluble material is present after sample homogenization with TRIzol.
This is often due to cell or tissue debris, which can hinder efficient RNA recovery. If insoluble material remains after homogenization and 5-minute room temperature incubation, centrifuge at 12,000 × g for 10 minutes at 4°C before chloroform addition. A clear supernatant and gelatinous pellet should appear. Carefully transfer the supernatant to a new tube and continue with the protocol.
3. Abnormal color in the aqueous phase after phase separation (e.g., yellow-brown, light pink, reddish).
Figure 2: Abnormal color in the aqueous phase after phase separation
This may be sample-specific:
- Skin tissue: Rich in lipids and melanin. Lipid droplets may carry pigment, causing colored aqueous layers. Centrifuge the homogenate before chloroform extraction to remove lipid layers from the top.
- Blood-rich samples: Hemoglobin may cause turbidity or yellowing in the aqueous phase. Iron in hemoglobin may also darken the organic phase if centrifugation is not performed at low temperatures. Pre-wash samples with PBS to reduce blood contamination.
- Insufficient TRIzol volume: If the sample-to-TRIzol ratio exceeds 1:10, particularly with fluid samples like blood, premature phase separation can occur, increasing DNA contamination. Add additional TRIzol or switch to a TRIzol formulation designed for liquid samples.
- High salt or protein content: Similar to above, this can also induce early separation. Pre-wash samples and/or increase TRIzol volume.
4. No white interphase appears after chloroform addition and centrifugation.
Figure 3: No white interphase appears after chloroform addition and centrifugation.
Possible causes:
- Insufficient mixing: After adding chloroform, vortex thoroughly. Before centrifugation, the mixture should appear milky. If it is clear, remix and centrifuge again.
- Low sample input: If the sample amount is small, the interphase may be faint or absent. Proceed with caution, and consider using a carrier such as glycogen to enhance RNA precipitation.
5. Precipitate forms at the bottom of the tube after centrifugation but before isopropanol addition.
These are likely polysaccharides or cell membrane residues. In samples with high blood content (e.g., liver), a reddish sticky layer may also appear. This layer contains blood-derived components and should be discarded. Only retain the aqueous phase above.
6. Abnormal color or consistency of RNA pellet.
Figure 4: Abnormal color or consistency of RNA pellet after mRNA extraction
Normally, RNA pellets appear clear or white. Unusual colors or textures (e.g., brown, gray, gelatinous) can result from:
-
Sample composition:
- Insects: Contain chitin, wax, or polysaccharides that cause brown precipitates.
- Polyphenol/polysaccharide-rich tissues: May result in colored or gel-like pellets. Reduce input amount and consider adding PVP during grinding or switch to a kit designed for such tissues.
- Refer to Issue 3 if colored aqueous phase was already observed.
- Operational error: Accidental aspiration of interphase/organic layer can contaminate RNA. Re-extract the aqueous phase with chloroform to purify.
7. No visible RNA pellet after precipitation.
Possible reasons:
- Low RNA content: Precipitate at 4°C or −20°C for 10–30 minutes after adding isopropanol. If a pellet is still not visible, avoid decanting; use pipetting to remove supernatant to prevent loss of invisible pellet. Add a carrier (e.g., glycogen or linear polyacrylamide) to improve RNA recovery in low-input samples. Salmon sperm DNA can also be used in some cases.
- Improper ethanol wash: If ethanol concentration is too low, RNA may dissolve or be lost. Ensure ethanol is freshly prepared with nuclease-free water.
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