3 Cell Line Extraction Methods

3.1 RNA

The night before, plate cells in a 6 well plate. 200K cells/well is a good number.

While one well is technically sufficient, performing in at least duplicate is prudent in case one sample doesn’t turn out after the extraction.

The following method is adapted from the mirVana miRNA Isolation Kit protocol

  • Get a bucket of ice
  • Set the heatblock to \(95^\circ C\) and add a microfuge tube with nuclease free H2O. You will need at least 50uL per sample - be generous with this!
  • For each sample, label 4 tubes - two standard 1.5mL microfuge tubes, two collection tubes

3.1.1 Lysing Cells

  1. Take 6 well plate from incubator.
  2. Aspirate off media, then gently add \(2mL\) PBS/well.
  3. Aspirate off PBS, then add \(300\mu L\) lysis buffer/well
  4. Incubate at RT for \(3\ minutes\)
  5. Use a cell scraper to scrape cells from the bottom of the well
    • Don’t forget to get all the edges
    • Try not to let the scraper skip
    • After scraping the bottom of the well, tilt the plate and sweep the lysate to the bottom to allow for easy transfer.
    • Use a new scraper for each well
  6. Transfer lysate to regular 1.5mL microfuge tube
  7. Put on ice

3.1.2 Organic Extraction

  1. Add \(\frac{1}{10}vol\) miRNA Homogenate Additive
  2. Vortex \(15\ seconds\)
  3. Put on ice for \(10\ minutes\)
  4. Add \(1\ vol\) phenol

‘1 volume’ refers to the pre-miRNA-Homogenate-Additive volume

  1. Vortex for \(45\ seconds\)
  2. Centrifuge at \(max\ speed\) for \(5\ minutes\)
  3. Put phenol away
  4. Remove upper phase, transfer to fresh 1.5mL microfuge tube

Err on the side of caution to avoid disturbing lower phase. As a rule of thumb, If you add \(300\mu L\) lysis buffer, remove roughly \(200\mu L\). Your mileage may vary.

Make sure to note the volume of upper face removed.

3.1.3 Total RNA Isolation

Spin = \(30\ seconds\) at \(10000 \times g\)

  1. Add \(1.25\ vol\) 100% EtOH
  2. Vortex \(15\ seconds\)
  3. Spin up to \(600\mu L\) sample through filter cartridge into collection tube
  4. Aspirate flow through
  5. Add \(600 \mu L\) Wash Solution 1, spin, aspirate, spin
  6. Add \(500 \mu L\) Wash Solution 2/3, spin, aspirate, spin
  7. Add \(500 \mu L\) Wash Solution 2/3, spin, aspirate, spin \(1\ minute\)
  8. Transfer filter to fresh collection tube
  9. Add \(50 \mu L\) \(95^\circ C\) H2O, spin
  10. Measure 260/280 and 260/230 via NanoDrop

3.1.4 TURBO DNA-free Treatment

Catologue Number: AM1907 Publication Number: 1907M Revision: H

  • Remove reagents from refrigerator, leave on ice.
  • Turn heat block to \(37^\circ C\)
  • Label one set of 1.5mL microfuge tubes, another set of 0.5mL microfuge tubes.
  • If you have many samples, a 96 well plate (with V bottoms) may be a better idea.
  • You should still prepare a set of labelled tubes for storage
  • If RNA concentration \(>200ng/\mu L\), dilute to \(200ng/\mu L\)

Anywhere between \(10-100\mu L\) sample is typical, with \(50\mu L\) being typical

  1. Add \(0.1\ volume\) of 10X TURBO DNase Buffer to RNA
  2. Add \(1\mu L\) TURBO DNase to RNA
  3. Mix gently
  4. Incubate at \(37^\circ C\) for \(30\ minutes\)
  5. Vortex DNase Inactivation Reagent to resuspend, then add \(0.1\ volume\) (minimum \(2\mu L\))

If the reagent is dried up and can’t be pipetted, determine how much volume of reagent there is, then add 25% of that volume of nuclease-free water.

  1. Incubate at \(RT\) for \(5\ minutes\), flicking every \(1.5\ minutes\)
  2. Centrifuge at \(10,000 \times g\) for \(1.5\ minutes\) for 1.5mL tubes, or \(2000 \times g\) for \(5\ minutes\) for 96-well plates.
  3. Transfer supernatant to labelled tube for storage. Store at \(\le -20^\circ C\)

Ensure you do not get any of the pellet.

3.2 DNA

The following method is adapted from a modified version of the DNeasy Blood & Tissue Kit (April 2016) protocol. This assumes you are using cell lines.

  1. Centrifuge \(\le 5\times10^6\) cells for \(5\ minutes\) at \(300 \times g\)
  2. Remove supernatant
  3. Add \(200\mu L\) PBS
  4. Add \(20\mu L\) proteinase K
  5. Add \(20\mu L\) 5mg/mL RNAse A. Incubate for \(5\ minutes\) at RT.
  6. Add \(200\mu L\) Buffer AL
  7. Add \(200\mu L\) 100% ethanol. Vortex thoroughly.
  8. Transfer solution to DNeasy Mini spin column place in a 2mL collection tube.
  9. Spin for \(1\ minute\) at \(\ge 6000 \times g\)
  10. Discard collection tube and replace with a fresh one.
  11. Add \(500\mu L\) Buffer AW1
  12. Spin for \(1\ minute\) at \(\ge 6000 \times g\)
  13. Discard collection tube and replace with a fresh one.
  14. Add \(500\mu L\) Buffer AW2
  15. Spin for \(3\ minutes\) at \(20000 \times g\)
  16. Discard collection tube and replace with a permanent collection tube.
  17. Add \(40\mu L\) nuclease-free H2O
  18. Incubate for \(1\ minute\)
  19. Spin for \(1\ minute\) at \(\ge6000 \times g\)

3.3 Protein

See Obtaining Protein Lysates