RNA Extraction Solution Trizol

Product Introduction

Product Information：

Total RNA Extraction Reagent is a broad-spectrum reagent designed for total RNA isolation. This reagent is suitable for extracting total RNA from animal tissues, plant materials, various microorganisms, and cultured cells. TRIzol effectively lyses samples while maximizing the preservation of RNA integrity. After adding chloroform and centrifugation, the mixture separates into three layers, with RNA distributed in the upper aqueous phase. The aqueous phase is collected, and total RNA is recovered via isopropanol precipitation.

It can be used in various molecular biology experiments, such as RT-PCR, Real-Time PCR, Northern blot, Dot blot, poly A selection, in vitro translation, and cDNA library construction.

Product Features:

1. RNase-Free Design: The complete kit is treated to be RNase-free, ensuring contamination-free operation.

2. Broad Applicability: Suitable for RNA extraction from various sample types including animal tissues, plant tissues, diverse microorganisms, and cultured cells.

3. User-Friendly Protocol: Simple and efficient procedure, with extraction completed in approximately 30 minutes.

4. High-Quality Output: Effectively preserves RNA integrity while delivering high yield and purity, free from protein or other contaminants. The extracted RNA typically exhibits an OD260/OD280 ratio of 1.9–2.2, making it ideal for a wide range of molecular biology applications.

Protocol: (Please read the precautions before starting the experiment)

Sample Processing:

1. Plant Tissue: Grind fresh plant tissue thoroughly in liquid nitrogen, or cut the tissue into small pieces and grind directly in TRIzol. Add 1 ml of TRIzol per 50–100 mg of tissue and mix well. Note: The sample volume should generally not exceed 10% of the TRIzol volume.

2. Animal Tissue: Cut fresh or -80°C frozen animal tissue into fine pieces. Add 1 ml of TRIzol per 30–100 mg of tissue and homogenize using a homogenizer. Alternatively, grind the tissue into powder in liquid nitrogen, transfer to a new centrifuge tube, and add 1 ml of TRIzol for mixing. Note: The sample volume should generally not exceed 10% of the TRIzol volume.

3. Adherent Cells: Directly lyse cells in the culture dish by adding TRIzol. Add 1 ml of TRIzol per 10 cm² of culture area and pipette repeatedly until no visible cell clumps remain. Insufficient TRIzol may lead to DNA contamination.

4. Suspended Cells: Centrifuge to collect cells. Add 1 ml of TRIzol per 5–10×10⁶ animal, plant, or yeast cells, or per 1×10⁷ bacterial cells, and mix well. Pipette repeatedly until no visible cell clumps remain. Do not wash the cells to avoid mRNA degradation.

5. For bacterial RNA extraction: Use the SpinPure™ Bacterial RNA Rapid Extraction Kit (Spin Column) (Cat. No.: 2120).

For blood or liquid sample RNA extraction: Use the TRI LS Reagent Liquid Sample Total RNA Extraction Reagent (TRIzol LS Reagent) (Cat. No.: 2107).

Extraction Procedure:

1. Allow the processed sample to stand at room temperature for 5–10 minutes to ensure complete dissociation of nucleoprotein complexes.

2. Optional step: Centrifuge at 12,000 rpm and 4°C for 10 minutes, then transfer the supernatant to a new RNase-free centrifuge tube. (Additional separation may be required for samples rich in proteins, fats, polysaccharides, or extracellular materials, such as muscle, adipose tissue, or plant tuber parts.)

3. Add 200 µl of chloroform per 1 ml of TRIzol. Vortex vigorously for 15 seconds and let stand at room temperature for 3 minutes.

4. Centrifuge at 12,000 rpm and 4°C for 10 minutes. The sample will separate into three layers: a lower organic phase, an intermediate protein layer, and an upper aqueous phase. RNA resides in the upper aqueous phase.

5. Transfer the aqueous phase to a new centrifuge tube and add an equal volume of isopropanol. Mix by inverting and let stand at room temperature for 10 minutes. (Avoid aspirating any precipitate or floating debris. Floating material may adhere to the outside of the pipette tip; ensure it is not transferred to the tube.)

6. Centrifuge at 12,000 rpm and 4°C for 10 minutes, then discard the supernatant. A gel-like pellet should form at the bottom of the tube.

7. Wash the pellet with 1 ml of 75% ethanol per 1 ml of TRIzol used, and vortex to resuspend.

8. Centrifuge at 12,000 rpm and 4°C for 3 minutes, then discard the supernatant (do not disturb the RNA pellet). For any residual liquid, briefly centrifuge again and carefully aspirate with a pipette.

9. Air-dry the pellet at room temperature for 2–3 minutes. Add 30–100 µl of RNase-free H₂O to fully dissolve the RNA. Avoid overdrying the pellet, as this may hinder dissolution. The eluted RNA can be used immediately or stored at –80°C.