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interactome:trizol [2019/12/05 11:20]
dan
interactome:trizol [2019/12/05 11:50]
dan
Line 25: Line 25:
  
  
-  * Freshly prepared DNaseI solution included in the kit+####Input sample requirements
  
-^  Check  ^  Reagent ​            ​^ ​  ​^ ​ 1 reaction (µL)  ^  _ _ _ _ reaction (µL)  ^ Memo                                              ^ +    ​IMPORTANT! 
-|         ​| ​ ddH2O (RNAse free)  |   ​| ​ 87.5                 ​| ​                        ​| ​                                                  | +    ​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.
-|         | 10x DNase Buffer ​    ​|   ​| ​ 10               ​| ​                        ​| ​                                                  | +
-|         ​| ​ DNaseI ​             |   ​| ​ 2.5          |                         ​| ​   12 units / µL                                               | +
-^         ​^ ​ Total volume (µL)   ​^ ​  ​^ ​ 100              ^                         ​^ ​ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  ^+
  
-\\ + 
-\\  + 
-\\ +^ Sample type                ^ Starting material per 1 mL of TRIzolTM Reagent ​                                                 ^ 
-\\  +| Tissues[1] ​                | 50 ー 100 mg of tissue ​                                                                            | 
-\\ +| Cells grown in monolayer ​  | 1 × 10xx5 ー 1 × 10xx7 cells grown in monolayer in a 3.5–cm culture dish (10 cm2)                  | 
-\\ +| Cells grown in suspension ​ | 5 ー 10 × 10xx6 cells from animal, plant, or yeasty origin or 1 × 10xx7 cells of bacterial origin ​ | 
 +|                            | [1] Fresh tissues or tissues stored in RNAlaterTM Stabilization Solution (Cat. No. AM7020). ​    | 
 +|                            |                                                                                                 | 
 +|                            |                                                                                                 | 
 +|                            |                                                                                                 | 
 + 
 + 
 +####​Procedural guidelines 
 +  
 +  *Perform all steps at room temperature (20–25°C) unless otherwise noted. 
 +  *Use cold TRIzolTM Reagent if the starting material contains high levels of RNase, such as spleen or pancreas samples. 
 +  *Use disposable, individually wrapped, sterile plastic ware and sterile, disposable RNase-free pipettes, pipette tips, and tubes. 
 +  *Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials. 
 +  *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.
 \\ \\
 \\ \\
Line 44: Line 55:
 #### Protocol #### Protocol
  
-==1  ​Sample preparation== +==1  ​Lyse samples and separate phases== 
-``` + 
-First step of sample ​preparation differs if you are using (A)culture cells OR (B)Tissue specimen.  +  *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material. 
-Buffers are highlighted ​in blue to prevent mistakes+ 
-``` +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:​ 
 +  *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
 +  *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.** 
 + 
 +    STOPPING POINT 
 +    Samples can be stored at 4°C overnight or at – 20°C for up to a year. 
 + 
 + 
 + 
 +  *(OptionalIf 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.txt
  • Last modified: 2019/12/05 11:51
  • by dan