Extraction of DNA, RNA, and Proteins from hiPSCs

This protocol allows simultaneous extraction of DNA, RNA, and proteins from cryopreserved hiPSC vials for downstream multi-omics analysis. The protocol uses Qiagen AllPrep kits with minimal modifications and has been verified in over 700 hiPSC lines in collaboration with Jeffrey Zhang, David Paik, and Yan Zhuge at the Joseph Wu lab at Stanford Cardiovascular Institute. For details, please see our publication on Current Protocols.


Extraction of DNA, RNA, and Proteins from hiPSCs

  • hiPSCs

    Thaw cells

    2 min
    1. Retrieve from cryostorage preserved hiPSC vials containing 1–2 million live cells, approximately the equivalent of a 6-well of 80% confluent cells.
    2. Thaw the cells gently in a water bath at 37 °C with stirring for 2 min, then transfer the cells to a 15 mL conical tube.
    3. Mix with 9 mL DMEM and spin at 300 g for 3 min at RT.
    4. Aspirate media and resuspend pellet in 1 mL DMEM.
    5. Count total live cells, e.g., using a Countess II FL Automated Cell Counter.
    6. Spin at 300 g 3 min at RT and aspirate media.
  • RNA

    Extract RNA

    20 min
    1. (Kit Instruction) Transfer 350 µL of QIAGEN RLT Buffer to each cell sample, then vortex well for ≥30 seconds to disrupt the cells and release molecules.
    2. Place the suspension in a Qiagen QIAShredder spin column and spin at 16,100 g for 2 min at RT to homogenize the cells, then follow the Qiagen AllPrep protocol to extract DNA.
    3. (Kit Instruction) Transfer the lysate to a QIAGEN AllPrep DNA spin column. Spin at 8,000 g for 30 s at RT. Keep the column for DNA extraction later.
    4. (Kit Instruction) Add 250 µL ethanol to the flowthrough , mix, and transfer to a QIAGEN RNeasy spin column. Spin at 8,000 g for 30 s at RT. Save the flowthrough of this step for protein extraction.
    5. (Kit Instruction) Add 700 µL Buffer RW1 and spin at 8,000 g for 30 s at RT.Remove flowthrough.
    6. (Kit Instruction) Add 500 µL Buffer RPE and spin at 8,000 g for 30 s at RT. Remove flowthrough.
    7. (Kit Instruction) Add 500 µL Buffer RPE and spin at 8,000 g for 2 min at RT. Remove flowthrough.
    8. (Kit Instruction) Spin the column at 16,100 g for 1 min at RT to dry. On a new set of tube, elute RNA yield by adding 55 µL RNAse-free water and spinning at 8,000 g for 1 min at RT. Repeat this step if necessary to improve yield.
    9. Quantify RNA yield, e.g., using a Thermo QuBit spectrofluorometer. Store by freezing on dry ice and transferring to –80°C cryostorage until use.
  • DNA

    Extract DNA

    10 min
    1. (Kit Instruction) To the DNA extraction column from the previous step, add 500 µL Buffer AW1. Spin at 8,000 for 30 s at RT.
    2. (Kit Instruction) Add 500 µL Buffer AW2. Spin at 8,000 for 30 s at RT. Transfer the DNA spin column to a new collection tube.
    3. (Kit Instruction) Heat the EB buffer to 55°C and add 55 µL to the center of the spin column membrane. Incubate for 2 min at RT.
    4. Spin at 8,000 g for 1 min at RT to elute DNA yield.
    5. Quantify DNA yield, e.g., using a Thermo QuBit spectrofluorometer, and assess gDNA integrity using a DNA gel. Store by freezing on dry ice and transferring to –80°C cryostorage until use.
  • Protein

    Extract Protein

    25 min
    1. (Kit Instruction) Add 600 µL Buffer AP to the previously saved protein flowthrough.
    2. Vortex very vigorously for up to 30 s at RT and incubate at RT for up to 10 min to precipitate proteins.
    3. Spin at 16,100 g for 10 min at 4°C. Aspirate supernatant, air dry the pellet, resuspend using common protocols for acetone precipitation or store at –80°C until use.

Materials

Example Outcome

The below plots show typical yields from 1-2 cryopreserved vials of hiPSCs from over 700 individual cell lines.

Lau Lab

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