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RNA exists in biological cells and some viruses and viroids, and RNA and protein levels need to be detected after cell transfection.

RNA extraction is the first step in RT-qPCR experiments, and its quality directly determines the results of RT-qPCR experiments. RNA is extracted by disrupting cells or tissues with denaturants, then extracting RNA through organic solvents such as chloroform, then precipitating, washing, drying, and finally dissolving.

The basic principle of RNA extraction is to use guanidineisothiocyanale as an effective decoupling agent, and when the cell is dissolved by it, the secondary structure of the protein disappears, the cell structure is degraded,then the nuclear protein quickly dissociates from the nucleic acid. In the presence of 4 mol/L guanidine isothiocyanate and β-mercaptoethanol, RNases are inactivated and intact total RNA can be extracted.

Below are some methods for extracting and detecting RNA from tissue for reference.

sample

Select cells in the vigorous growth period, select cells about 48 hours after treatment, if it is a tissue sample, try to choose fresh tissue sample, and it is better to extract RNA immediately after getting the sample.

Reagents and Instruments Required :

Reagent:

● Tissue

●Trizol reagent

●Chloroform

●Isopropanol

●75% ethanol (prepared with RNase-Free water)

● RNase-Free water

● Liquid nitrogen

Instruments &Consumables

●1.5 ml Micro tubes (RNA-free);

●Pipette Tips (RNA-free);

● Micro Pipettes

● Vortex shaker

● High-speed refrigerated centrifuge

● Ultra-clean bench

● Homogenizer or grinder

Experimental Procedure

● Cut the tissue into 1-3 mm pieces and put into 1.5mL micro tube, add 0.5-1 mL of Trizol lysate (liquid nitrogen grinding is recommended for some harder tissues), and rapidly grind by using a liquid nitrogen grinder. Add 1/5 volume of chloroform to the micro tube, mix well with vigorous shaking, and wait for 10 minutes to completely dissociate the nucleic acid protein complex.

● Using a high-speed refrigerated centrifuge, centrifuge at 12,000 rpm at 4°C for 15 minutes.

● After centrifugation, carefully transfer the upper aqueous phase (approximately 1/2 volume of Trizol) to a new micro tube, add an equal volume of isopropanol, and wait for 5 to 10 minutes after shaking.

● Continue centrifuge at 12,000 rpm at 4°C for 15 minutes and keep the sediments.

● Add 1mL of 75% ethanol to wash and precipitate RNA, and continue centrifuge at 4°C 12000rpm for 10 minutes.

● Dispose the supernatant and repeat once.

● Open the EP tube cap in a clean bench, and allow to air dry for 5-10 minutes without complete drying.

● Depending on the amount of precipitate, add 10-30 μL of RNase-Free DEPC double distilled water.

● After the precipitate is dissolved, its concentration and purity are determined and labeled with nanodrop for further experiments.

Experimental results and analysis

● Take 4 μL of RNA sample, dilute 100 times, and measure absorbance values at 260 nm and 280 nm on a spectrophotometer.

● Sample RNA concentration (μg/mL) = A260 x 40 (μg/mL) x dilution. A260/A280 should be between 1.9~2.0, if it is less than this value, there may be protein impurities.

Experimental Notes

● When extracting total RNA, it is necessary to pay attention to prevent contamination of RNases. Glassware, saliva and sweat in the general laboratory may be the source of RNase contamination, so disposable gloves should always be worn during operation, glassware should be dried at 250 °C for more than 4 hours or 180 °C for more than 9 hours. Other utensils such as electrophoresis apparatus should be soaked in 3% H2O2 for more than 2 hours, rinsed with autoclaved distilled water treated with 0.2% DEPC for more than 3 times, and dried for later use.

● Solutions should be prepared using RNASE-free water (add water to a clean glass bottle, add DEPC to a final concentration of 0.1% (V/V), keep it overnight and autoclave it. Note: DEPC is suspected to be carcinogenic, so it should be handled with care)

● Trizol lysate is very dangerous, so be careful not to splash on your body, and clean Trizol on the countertop in time.

The above are some experimental procedures and precautions, Welso is a global supplier to provide laboratory consumables and equipments, welso can provide, such as high-speed refrigerated centrifuges, vortex shakers, grinders, etc., which are required in such experiment. If you need to order these equipment, you can contact at any time, we will reply you within 24 hours.