Trizen RNA Plus is a reagent designed for efficient and rapid isolation of total RNA from animal or plant tissues as well as cultured cells. To use it, first homogenize the tissues or cells in Trizen RNA Plus solution. Next, add chloroform to the homogenate, mix thoroughly, and then centrifuge the mixture. This centrifugation step separates the solution into three distinct layers:

1. The top layer consists of a clear liquid containing the isolated RNA.
2. The middle layer comprises a semi-solid material containing DNA.
3. The bottom layer is a red-colored organic solvent containing proteins, polysaccharides, fatty acids, cell debris, and a small amount of DNA.

This process effectively isolates the desired RNA from the biological sample, with the various components of the sample partitioning into distinct layers based on their physical properties.

After removing the top clear liquid layer containing the isolated RNA and transferring it to a new tube, take care not to disturb the middle DNA-containing layer. Proceed with an isopropanol precipitation step to extract the total RNA efficiently.

The resulting isolated total RNA is of high quality, free from contaminants such as DNA or proteins, making it suitable for a variety of downstream applications including RT-PCR, Northern blot analysis, mRNA isolation, and in vitro translation reactions.

The products are intended solely for research purposes and should not be utilized for diagnostic assays.

• The product can be stored is stored at room temperature (24⁰C) for up to 2 years

RNA Isolation Protocol

Adherent cells

• Remove the media from the cells and wash with 1X PBS
• Add 1 – 2 ml of Trizen RNA Plus directly onto the 10 cm² petri dish containing adherent cells. Swirl the reagent around the plate to ensure thorough coverage of the cell surface with Trizen RNA Plus.
• Use a pipette to collect the cells along with the Trizen RNA Plus solution and transfer them into a centrifuge tube. Pipette the solution up and down several times to ensure complete resuspension of the cells.
• Allow the samples to incubate at room temperature for 5 minutes. Non-adherent cells
• Centrifuge the cells and media at 8,000g for 2 minutes at 4℃. Carefully discard the supernatant without disturbing the cell pellet.
• Add 1 ml of Trizen RNA Plus for every 5 x 10^6 cells.
• Pipette the solution up and down until the cell pellet is completely resuspended.
• Allow the mixture to incubate at room temperature for 5 minutes to facilitate the isolation of RNA from nuclear proteins.

Animal and plant tissue sample

• Immediately transfer frozen tissue into a mortar, add liquid nitrogen, and crush with a pestle until the tissue is homogenized into a powdery consistency. Ensure thorough homogenization as incomplete homogenization may impact RNA quality or yield.
• Add an appropriate volume of Trizen RNA Plus corresponding to the amount of tissue homogenized. For fresh tissue, add Trizen RNA Plus immediately after collection and homogenize completely.
• Transfer the homogenized sample into a centrifuge tube and let it stand at room temperature for 5 minutes.
• Centrifuge the tube at 12,000g for 5 minutes at 4℃.
• Carefully collect the supernatant by pipetting and transfer it to a new centrifuge tube, taking care not to disturb the pellet.

Extracting total RNA

• To the solution obtained from the previous steps, add 0.2 ml of chloroform per 1 ml of Trizen RNA Plus used for homogenization. Cap the centrifuge tube and vigorously mix until the solution appears milky.
• Allow the mixed solution to stand at room temperature for 5 minutes.
• Centrifuge the tube at 12,000rpm for 15 minutes at 4℃. Following centrifugation, the solution will separate into three distinct layers: a top liquid layer containing RNA, a semi-solid middle layer consisting mostly of DNA, and a bottom organic solvent layer.
• Carefully transfer the top liquid layer (containing RNA) to a new centrifuge tube without disturbing the middle DNA- containing layer.
• To the transferred RNA-containing solution, add 0.5 – 1 ml of isopropanol per 1 ml of Trizen RNA Plus used for homogenization. Mix the solution thoroughly and then let it stand at room temperature for 10 minutes.
• Centrifuge the tube at 12,000rpm for 10 minutes at 4℃ to precipitate the RNA. The RNA will appear as a pellet at the bottom of the tube after centrifugation.
• Carefully remove the supernatant from the tube without disturbing the RNA pellet. It is okay if some residual isopropanol remains.
• Add an equivalent volume of 75% cold ethanol to the volume of the supernatant that was removed. Gently vortex the solution to wash the RNA pellet.
• Centrifuge the tube at 8,000rpm for 5 minutes at 4℃ to pellet the RNA again.
• Discard the supernatant by carefully pouring it off, taking care not to disturb the RNA pellet.
• Dry the RNA precipitate by allowing the tube to remain open for several minutes.
• Once the precipitate is dry, carefully dissolve it in an appropriate volume of RNase-free water (50 – 100 µL). Avoid using centrifugation or heat to dry the precipitate, as this may impede the dissolution of the RNA.