• Total RNA Isolation Reagent

Total RNA Isolation Reagent Quick Details

  • CAS No.: no CAS number
  • Place of Origin: China (Mainland)
  • Grade Standard: Reagent Grade
  • Appearance: transparent
  • Application: RNA isolation

1. Sample Preparation

 1A. Tissue: Homogenize tissue samples in RNApro reagent (1 ml per 50–100 mg of tissue) 

in an appropriate homogenizer. The volume of the tissue should not exceed 10% of the volume of the RNApro reagent. 

 1B. Monolayer cells: Lyse cells directly on the culture dish. Use 1 ml of the RNApro reagent per 10 cm2 of culture plate surface area. After addition of the reagent, the cell lysate should be passed several times through a pipette to form a homogenous lysate.  

 1C. Suspension cells: Isolate cells by centrifugation and then lyse in RNApro reagent by repeated pipetting.  One ml of the reagent is sufficient to lyse 5–10×106 animal, plant, or yeast cells, or 107 bacterial cells. 

 

2. Phase Separation

To ensure complete dissociation of nucleoprotein complexes, allow samples to stand for 5 minutes at room temperature. Add 0.1 ml of 1-bromo-3-chloropropane or 0.2 ml of chloroform per ml of RNApro reagent used. Cover the sample tightly, shake vigorously for 15 seconds, and allow to stand for 2–15 minutes at room temperature. Centrifuge the resulting mixture at 12,000×g for 15 minutes at 2-8 °C. 

Centrifugation separates the mixture into 3 phases: a green organic phase (containing protein), an interphase (containing DNA), and a colorless upper aqueous phase (containing RNA). 

RNA Isolation

1. Transfer the aqueous phase to a fresh tube and add 0.5 ml of 2-propanol per ml of RNApro reagent used in Sample Preparation, step 1 and mix. Allow the sample to stand for 5–10 minutes at room temperature. Centrifuge at 12,000×g for 10 minutes at 2-8 °C. The RNA precipitate

 will form a pellet on the side and bottom of the tube. 

Note: To minimize small RNA (eg. miRNA) loss, substitute the 2-propanol by 2.5-volume of ethanol and store the sample at -20 °C for 30 minutes. 

2. Remove the supernatant and wash the RNA pellet by adding a minimun of 1 ml of 75% ethanol. Vortex the sample and then centrifuge at 7,500×g for 5 minutes at 2-8 °C. Samples can be stored in ethanol at 2-8 °C for at least 1 week and up to 1 year at –20 °C. 

3. Vacuum or air dry the RNA pellet for 5–10 minutes. Do not allow the RNA to dry completely, because the pellet can lose solubility.

4. Resuspend the RNA pellet in formamide, RNase-free water or 0.5% SDS solution (20–50 μL) by passing the solution up and down several times through a pipette tip. Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions. To facilitate dissolution, mix by repeated pipetting with a micropipette at 55–60 °C for 10–15 minutes 

5. Proceed to downstream application, or store at –70°C. 

DNA Isolation

1. Carefully remove the remaining aqueous phase overlaying the interphase and discard. To precipitate the DNA from the interphase and organic phase, add 0.3 ml of 100% ethanol per 1 ml of RNApro reagent used in Sample Preparation, step 1. Mix by inversion and allow to stand for 2–3 minutes at room temperature. Centrifuge at 2,000×g for 5 minutes at 2-8 °C. Note: Removal of the remaining aqueous phase before DNA precipitation is a critical step for the quality of the isolated DNA. 

2. Remove the supernatant and save at 2-8 °C for protein isolation. Wash the DNA pellet twice in 0.1 M trisodium citrate, 10% ethanol solution. Use 1 ml of wash solution for every 1 ml of RNApro reagent used in Sample Preparation, step 1. During each wash, allow the DNA pellet to stand (with occasional mixing) for at least 30 minutes. Centrifuge at 2,000×g for 5 minutes at 2-8 °C. Resuspend the DNA pellet in 75% ethanol (1.5–2 ml for each ml RNApro reagent) and allow to stand for 10–20 minutes at room temperature. 

3. Dry the DNA pellet for 5–10 minutes under a vacuum and dissolve in 8 mM NaOH with repeated slow pipetting with a micropipette. Add sufficient 8 mM NaOH for a final DNA concentration of 0.2–0.3 mg/mL (typically 0.3–0.6 ml to the DNA isolated from 50–70 mg of tissue or 107 cells). This mild alkaline solution assures complete dissolution of the DNA pellet. Centrifuge at 12,000×g for 10 minutes to remove any insoluble material and ransfer the supernatant to a new tube. 

Protein Isolation

1. Precipitate proteins from the phenol-ethanol supernatant (DNA Isolation, step 2) with 1.5 ml of  2 propanol per 1 ml of RNApro reagent used in Sample Preparation, step 1. Allow samples to stand for at least 10 minutes at room temperature. Centrifuge at 12,000×g for 10 minutes at 2-8 °C.  

Note: For some samples, the protein pellet may be difficult to dissolve in 1% SDS (step 3). Use this   alternate procedure to correct the problem:  

a. Dialyze the phenol-ethanol supernatant against three changes of 0.1% SDS at 2-8 °C.  

b. Centrifuge the dialysate at 10,000×g for 10 minutes at 2-8 °C.  

c. The clear supernatant contains protein that is suitable for use in Western blotting procedures. 

2. Discard supernatant and wash pellet 3 times in 0.3 M guanidine hydrochloride/95% ethanol solution, using 2 ml per 1 ml of RNApro reagent used in Sample Preparation, step 1. During each wash, store samples in wash solution for 20 minutes at room temperature. Centrifuge at 7,500 ´ g for 5 minutes at 2-8 °C. After the 3 washes, add 2 ml of 100% ethanol and vortex the protein pellet. Allow to stand for 20 minutes at room temperature. Centrifuge at 7,500×g for 5 minutes at 2-8 °C. 

Note: Protein samples suspended in 0.3 M guanidine hydrochloride/95% ethanol solution or 100% ethanol can be stored for 1 month at 2-8 °C or 1 year at –20 °C. 

3. Dry protein pellet under a vacuum for 5–10 minutes. Dissolve pellet in 1% SDS aided by working the plunger of micropipette with tip in the solution. Remove any insoluble material by centrifugation at 10,000×g for 10 minutes at 2-8 °C. Transfer supernatant to a new tube. The protein solution should be used immediately for Western blotting or stored at –20 °C.

Total RNA Isolation Reagent

  • Product Code: WDRQ0001
  • Availability: In Stock
This product has a minimum quantity of 10

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Tags: DNA, Reagents