Extraction of DNA, RNA, and Proteins from hiPSCs

Extraction of DNA, RNA, and Proteins from hiPSCs

• hiPSCs

  Thaw cells

  Live Cell Counts
  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

  Solid Phase Extraction
  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

  Solid Phase Extraction
  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

  Protein Precipitation
  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.