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interactome:trizol [2019/12/05 11:31]
dan
interactome:trizol [2019/12/05 11:51] (current)
dan
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 | RNase-free water of 0.5% SDS         ​| ​      | | RNase-free water of 0.5% SDS         ​| ​      |
 | (Optional) RNase-free glycogen ​      ​| ​      | | (Optional) RNase-free glycogen ​      ​| ​      |
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 ####Input sample requirements ####Input sample requirements
-**IMPORTANT!** + 
 +    ​IMPORTANT!
     Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.     Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.
  
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   *Always use proper microbiological aseptic techniques when working with RNA.   *Always use proper microbiological aseptic techniques when working with RNA.
   *Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.   *Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.
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   *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.   *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.
- 
-Tissues: 
-  *Add 1 mL of TRIzolTM Reagent per 50–100 mg of tissue to the sample and homogenize using a homogenizer. ​ 
  
 Cell grown in monolayer: Cell grown in monolayer:
   *Remove growth media.   *Remove growth media.
   *Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.   *Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.
-  *Pipet the lysate up and down several times to homogenize. 
-  
-Cells grown in suspension: 
-  *Pellet the cells by centrifugation and discard the supernatant. 
-  *Add 0.75 mL of TRIzolTM Reagent per 0.25 mL of sample (5– 10 × 106 cells from animal, plant, or yeasty origin or 1 ×107 cells of bacterial origin) to the pellet. 
-**Note: Do not wash cells before addition of TRIzolTM Reagent to avoid mRNA degradation.** 
   *Pipet the lysate up and down several times to homogenize.   *Pipet the lysate up and down several times to homogenize.
  
  
 **Note: The sample volume should not exceed 10% of the volume of TRIzolTM Reagent used for lysis.** **Note: The sample volume should not exceed 10% of the volume of TRIzolTM Reagent used for lysis.**
 +
 +    STOPPING POINT
 +    Samples can be stored at 4°C overnight or at – 20°C for up to a year.
 +
 +  *(Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
 +  *Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
 +  *Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysis, then securely cap the tube.
 +  *Incubate for 2–3 minutes.
 +  *Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
 +  *The mixture separates into a lower red phenol-chloroform,​ and interphase, and a colorless upper aqueous phase.
 +  *Transfer the aqueous phase containing the RNA to a new tube. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
 +
 +    IMPORTANT! ​
 +    Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase. ​  
 +
 +
 +  *Proceed directly to “Isolate RNA“ ​
 +
 +\\
 +\\
 +==2  Isolate RNA==
 +
 +Precipitate the RNA
 +  *(Optional) If the starting sample is small (<106 cells or <10 mg of tissue), add 5–10 μg of RNase-free glycogen as a carrier to the aqueous phase.
 +Note: The glycogen is co-precipitated with the RNA, but does not interfere with subsequent applications.
 +  *Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzolTM Reagent used for lysis. Incubate for 10 minutes.
 +  *Centrifuge for 10 minutes at 12,000 × g at 4°C.
 +  *Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
 +  *Discard the supernatant with a micropipettor.
 +\\
 +\\
 +Wash the RNA
 +  *Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzolTM Reagent used for lysis.
 +Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
 +  *Discard the supernatant with a micropipettor.
 +  *Vacuum or air dry the RNA pellet for 5–10 minutes.
 +    IMPORTANT! ​
 +    Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure total solubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.
 +\\
 +\\
 +Solubilize the RNA
 +  *Resuspend the pellet in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
 +
 +    IMPORTANT! ​
 +    Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic reactions.
 +
 +  *Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes. ​
     ​     ​
 +
 +
 +\\
 +Proceed to downstream applications,​ or store the RNA at –70°C.
 +
 +\\
 +\\
 +\\
 + --- //​[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/12/05 11:39//
  • interactome/trizol.1575513115.txt.gz
  • Last modified: 2019/12/05 11:31
  • by dan