RNA extraction experimental

The essence of RNA extraction from different tissues is the process of lysing cells to release RNA, and removing impurities such as protein and DNA through different methods to finally obtain high-purity RNA products. The commonly used traditional method at present is through the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for various animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials. Trizol contains protein denaturants such as guanidinol isothiocyanate and β-mercaptoethanol, which can quickly break cells and effectively inhibit the RNase activity released by cells. By dissolving proteins, protein secondary structures disappear, cell structures are degraded, and proteins, DNA, RNA and other substances are released. Combined with density gradient ultracentrifugation, RNA is precipitated at the bottom of the tube, while DNA and protein are in the supernatant^[1][2][3].

MCE has not independently verified the accuracy of these methods. They are for reference only.

Extracting high-quality RNA from samples from various sources, such as bacteria, blood, animal tissues, and cultured cells, results in different pre-treatment methods due to differences in cell structure and composition.

• Attached cell

Discard the culture medium and wash 1-2 times with pre cooled 1 x PBS. Then add 1 mL of Trizol to every 10 cm² area of the culture plate to fully cover the cell surface. Use a pipette to blow several times to completely remove the cells. Then transfer the lysate to a 1.5 mL centrifuge tube, repeatedly blow and mix with a pipette to fully lyse, and let it stand on ice for 5 minutes.

(Note: The amount of Trizol added is determined by the area of the culture plate, not by the number of cells. If the dosage of Trizol is insufficient, it may lead to DNA contamination in the extracted RNA.)

• Suspension cell

Collect cells by centrifugation and wash 1-2 times with 1 x PBS. Add 1 mL of Trizol to every 5 × 10⁶-10⁷ cells (approximately 1 × 10⁸ CFU of bacteria), mix repeatedly with a pipette to fully lyse, and let stand on ice for 5 minutes.

• Organizational samples

Grind fresh tissue with liquid nitrogen until there are no obvious particles. Add 1 mL of Trizol to every 50-100 mg of animal tissue, and use a pipette to repeatedly blow and mix until fully lysed. Alternatively, a homogenizer can be used for homogenization treatment. Transfer the lysis solution to a centrifuge tube, centrifuge at 4 ° C 12000 g for 5 minutes, and aspirate the supernatant.

• Add 1/5 Trizol volume of pre-cooled chloroform to the above lysis solution, let stand the formed emulsion on ice for 5 min.

• Centrifuge at 12000g for 15 minutes at 4°C. At this time, the sample is divided into three layers: the upper layer is a colorless aqueous phase, the middle layer is a white DNA and protein layer, and the lower layer is a pink organic phase. RNA is mainly found in the upper layer.

• Remove the centrifuge tube carefully and pipette as much of the upper colorless aqueous phase into a new centrifuge tube as possible, about 600 μL, to avoid sucking the middle layer.

• Add an equal volume of pre-cooled isopropyl alcohol, mix by inverting, and let standing at -20°C for 10 minutes.

• Centrifuge at 12,000g for 10 minutes at 4°C. A white precipitate will usually be visible. Carefully discard the supernatant, add 1 mL of 75% ethanol prepared with pre-cooled DEPC water, flick the bottom of the tube to fully wash the precipitate, and let standing on ice for 5 minutes.

• Centrifuge at 12000 g for 5 min at 4°C, remove the supernatant, repeat step 5 and wash again. After discarding the supernatant, dry the pellet in a clean environment at room temperature for 5-10 minutes.

• Add 20-50 μL RNase-Free dd H2O or DEPC water to dissolve RNA, take a small amount for detection, and freeze the rest at -80°C.

• RNA purity can be tested with a spectrophotometer. RNA has a continuous absorption spectrum with the highest absorption peak at 260 nm; protein has the highest absorption peak at 280 nm. If impurities are present, such as ethanol, GTC, GuHCl, EDTA, etc., they can be observed at 230 nm. In general, a small amount of protein, salt or genomic contamination is acceptable. The OD₂₆₀/OD₂₈₀ ratio of the RNA sample is detected, which can directly reflect the purity of the RNA.

• Alternatively, 1% agarose gel electrophoresis can be used to check the integrity of the RNA. Taking mammalian cell tissue as an example, three clear bands can be seen in complete total RNA, with molecular weights of 28 S, 18 S, and 5 S respectively from top to bottom. If three bands can be seen, but the bands are blurred or diffuse, it means that the RNA has been partially degraded; if only one band with a very small molecular weight or no band can be seen, it means that the RNA has been completely degraded.

• RNA Concentration (ng/μL)= OD₂₆₀dilution factor × 40 or directly use NanoDrop instrument to detect.

1. Tissue preservation

• It is best to use fresh samples when extracting RNA. If RNA cannot be extracted in time, it should be quickly frozen in liquid nitrogen and then stored at -80°C to avoid repeated freezing and thawing, which will lead to degradation of the extracted RNA and reduction in extraction volume. It is not recommended to freeze tissue directly at -80°C because samples freeze slowly and endogenous RNase can cause RNA degradation during this process.

2. Low RNA purity

• The entire process must be conducted under RNase-free contamination conditions. All consumables such as pipette tips and centrifuge tubes used in experiments must be RNase-free. All related solutions must be prepared with RNase-free water or DEPC water. Gloves and masks must be worn during the experiment, and new gloves need to be replaced frequently. Because skin often contains bacteria, it may lead to RNase contamination, which degrades RNA and results in low RNA yield.

• When adding chloroform, mix vigorously to thoroughly mix the two phases. DNA will be retained in the organic phase under the acidic conditions of water-saturated phenol and will not escape into the aqueous phase. When aspirating the supernatant, try not to separate it into different new EP tubes. When the amount itself is not large, try to concentrate it into one tube. At the same time, avoid absorbing the intermediate protein and organic phase, thereby reducing the purity of the RNA.

• The final RNA pellet is not completely dissolved. If necessary, you can gently pipette it a few times with a pipette.

3. No precipitate after adding isopropyl alcohol.

• Normally, you can see a precipitate after adding isopropyl alcohol. If you don’t see an obvious white precipitate, it may be that the RNA content is low or the sample volume is too low. You can leave it at -20°C overnight and then centrifuge.

4. The extracted RNA is contaminated with DNA.

• Chloroform is miscible with water at a certain ratio at room temperature. Centrifugation at room temperature will cause a small amount of DNA contamination in the upper aqueous phase. Therefore, low-temperature high-speed centrifugation is required after adding chloroform. When sucking the upper layer liquid, try to avoid sucking into the middle layer and lower layer.

• Too little reagent added when homogenizing the sample.