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interactome:experiments:20200329 [2020/03/29 21:27]
dan created
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-## RNA extraction ​from HEK293T cells+## RNA extraction ​with TriZol and column kit (ZYMO RNA concentration kit 25)
 {{tag>​interactome PersonalInteractome}} {{tag>​interactome PersonalInteractome}}
 +
 +Based on the user guide of Trizol reagent by Invitrogen and Column kit (ZYMO).
  
 #### Material #### Material
-  * Sample : 6 x 10^6 of cell-line cells x 2 (with help from Arman) 
-  * ISOSPIN Cell & Tissue RNA kit (Nippon Gene, Cat.314-08211) 
-ISOSPIN Cell & Tissue RNA (For 50 reactions) 
-^ Components ​                                  ^ Volume ​               ^ Storage ​ ^ Memo                                       ^ 
-| PT Extraction Buffer(For Tissue) ​            | 30 ml x 1             | RT       ​| ​                                           | 
-| C Extraction Buffer(For cell) ​               | 30 ml x 1             | RT       ​| ​                                           | 
-| PT Binding Buffer(For both Tissue and cell) ​ | 40 ml x 1             | RT       | Includes EtOH                              | 
-| PT Wash1 Buffer ​                             | 40 ml x 1             | RT       | Includes EtOH                              | 
-| PT Wash2 Buffer ​                             | 40 ml x 1             | RT       | Includes EtOH                              | 
-| DNase I (RNase free)                         | 2,000 units x 1 tube  | -20°C ​   |                                            | 
-| 10 x DNase I Buffer ​                         | 1 ml x 1              | -20°C ​   |                                            | 
-| ddWater (RNase free)                         | 1 ml x 8              | RT       | For preparaing DNase I solution & elution ​ | 
-| Spin Column ​                                 | 50  columns ​          | RT       | Column + collection tube                   | 
  
 +####Input sample requirements
  
 +    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.
  
  
  
-  ​* Freshly prepared DNaseI solution included ​in the kit+^ 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). ​    | 
 +|                            |                                                                                                 | 
 +|                            |                                                                                                 | 
 +|                            |                                                                                                 |
  
-^  Check  ^  Reagent ​            ​^ ​  ​^ ​ 1 reaction (µL)  ^  2 reaction (µL)  ^ Memo                   ^ 
-|         ​| ​ ddH2O (RNAse free)  |   ​| ​ 87.5             ​| ​ 175              |                        | 
-|         | 10x DNase Buffer ​    ​| ​  ​| ​ 10               ​| ​ 20               ​| ​                       | 
-|         ​| ​ DNaseI ​             |   ​| ​ 2.5              |  5                | 30 units. 12 units/​µL ​ | 
-^         ​^ ​ Total volume (µL)   ​^ ​  ​^ ​ 100              ^  200              ^                        ^ 
  
-\\ +####​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.
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 #### 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.  +
-Buffers are highlighted in blue to prevent mistakes. +
-``` +
-   +
-**A) If you are using cell-line: ** +
-  *Collect at most 6 x 10^6 of cultured cell-line. +
-  *Add <color #​00a2e8>​600 µL of C Extraction Buffer (For cells)</​color>​ +
-  *Lyse cells by pipetting ​+
  
 +  *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.
  
-**B) If you are using Tissue samples** +Cell grown in monolayer
-  *Take a at most 20 mg of fresh or frozen Tissue sample in a 1.5mL tube +  *Remove growth media
-  *Add <color #​00a2e8>​600 µL of PT Extraction Buffer (For Tissue)</​color>​ +  *Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells. 
-  *Grind the Tissue specimen with a pestle +  *Pipet the lysate up and down several times to homogenize.
-\\ +
-\\ +
  
-**Following procedures are same for both Cell-line and Tissue samples.** 
  
-  ​*Vortex ​the samples ​for more than 30 secs+**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.
-==2  PT binding==+
  
-  *Centrifuge sample ​at 13,000 g, 10 mins, 4C  +  *(Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes ​at 12,000 × at 4–10°Cthen transfer the clear supernatant to a new tube
-  *To a new tube, transfer supernatant (*remember volume ​for next step* +  *Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex. 
-  *Add <color #​00a2e8>​PT binding buffersame volume as removed supernatant in previous step</​color>​ +  *Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysisthen securely cap the tube. 
-  *Mix by inverting ​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-chloroformand 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. 
 + 
  
-\\ 
-\\  
-==3  Column loading== 
-``` 
-Load the whole sample by repeating [loading -> centrifuge -> discard flow through] twice. 
-``` 
-  *Add 600 µL of the sample mixture to the column 
-  *Centrifuge sample at 13,000 x g, 1 min, 4C 
-  *Discard flow through 
-  *Add rest of sample mixture to the column ​ 
-  *Centrifuge sample at 13,000 x g, 1 min, 4C 
-  *Discard flow through 
- 
- 
-\\ 
-\\  
-==4  Wash and DNaseI treatment== 
- 
-  *Add <color #​00a2e8>​500 µL of PT wash buffer1</​color>​ to the column 
-  *Centrifuge sample at 13,000 x g, 1 min, 4C 
-  *Discard flow through 
- 
-  *Add <color #​00a2e8>​100 µL of DNaseI </​color>​ to the column 
-  *Incubate for 15 minute at room temprature 
- 
-  *Add <color #​00a2e8>​300 µL of PT wash buffer1</​color>​ to the column 
-  *Centrifuge sample at 13,000 x g, 1 min, 4C 
-  *Discard flow through 
- 
-  *Add <color #​00a2e8>​600 µL of PT wash buffer2</​color>​ to the column 
-  *Centrifuge sample at 13,000 x g, **2 min**, 4C 
-  *Discard flow through 
- 
- 
-\\ 
-\\  
-==5  Elution== 
- 
-  *Place the column on a new, labled 1.5 mL tube for collecting eluate 
-  *Add <color #​00a2e8>​50 µL of ddH2O</​color>​ to center of the membrane of the column 
-  *Incubate on bench for 3 mins 
-  *Centrifuge sample at 13,000 x g, 1 min, 4C 
- 
- 
-\\ 
-\\  
-==6  Quantification== 
- 
-  *Measure concentration of RNA sample 
  
 +== 2 RNA binding and DNaseI treatment==
  
- +  *Add same volume of EtOH to the aqueous phase from the previous step. 
 +  *Transfer tube to the Zymo spin IICR Column in a collection tube and centrifuge for 30 seconds (@12,000 xg). 
 +  *Discard flow-through 
 +  *Prewash the column with 400 µL of RNA Wash Buffer and centrifuge for 30 seconds (@12,000 xg).    
 +  *Discard flow-through 
 +  *To each reaction, add a Master mix containing 5 µL of DNaseI and 75 µL 1 x DNaseI buffer. 
 +  *Incubate at room temperature for 15 minutes. 
 +  *Add 700 µL of RNA Wash Buffer to the column and and centrifuge for 30 seconds (@12,000 xg).    
 +  *Add 400 µL of RNA Wash Buffer and centrifuge for 2 min (@12,000 xg) to remove all remaining buffer. ​   
 +  *Transfer column carefully to a new RNase-free tube. 
 +  *Add 50 µL of ddH2O to the column and centrifuge for 30 seconds (@12,000 xg).   
    
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  • interactome/experiments/20200329.1585484859.txt.gz
  • Last modified: 2020/03/29 21:27
  • by dan