User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/08

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Summary

To obtain RNA for Real Time PCR RNA needs to isolated, to compare this to changes seen in protein expression. For best comparison also protein can be isolated from the same sample.

Materials & Methods

Materials

Method

RNA isolation for Real time qPCR

Notes
  • For RNeasy column. Do not exceed RNA limit (100 µg), for unknown RNA content do not exceed 3–4×106 cells
  • Clean bench & pipette with RNAway
Procedure
  1. Per Ø10 cm dish collect cells in 3 mL TRIzol® Reagent
  2. Incubate 5’ @ RT
  3. Add chloroform, 0.2 mL/mL TRIzol® Reagent
  4. Vortex to mix
  5. Incubate 2–3’ @ RT
  6. Centrifuge 15’ 12.000×g @ 4°C
  7. Collect supernatant
    1. Save red, phenol/chloroform phase for DNA/Protein isolation?
  8. Add isopropanol, 0.5 mL / mL TRIzol® Reagent
  9. Incubate 10’ @ RT
  10. Centrifuge 10’ 12.000×g @ 4°C
  11. Remove supernatant and resuspend pellet in 500 µL/ mL TRIzol® Reagent 100% EtOH
  12. Transfer sample (<700 µL) to RNeasy spin column (w. collection tube)
  13. Centrifuge 15 s, ≥8000×g (≥10.000 RPM), discard flowthrough
    1. >700 µL of sample can be put through the same column (<100 µg RNA)
  14. Add 700 µL of Buffer RW1 to column
  15. Centrifuge 15 s, ≥8000×g (≥10.000 RPM), discard flowthrough
  16. Add 500 µL of Buffer RPE to column
  17. Centrifuge 15 s, ≥8000×g (≥10.000 RPM), discard flowthrough
  18. Add 500 µL of Buffer RPE to column
  19. Centrifuge 2 min, ≥8000×g (≥10.000 RPM), discard flowthrough
  20. OPTIONAL, Centrifuge 1 min, ≥8000×g (≥10.000 RPM), discard flowthrough
  21. Put column to new 1.5 mL tube, add 30–50 µL RNAse free water
  22. Centrifuge 1 min, ≥8000×g (≥10.000 RPM)
  23. Determine RNA concentration by Nanodrop
    1. @ >10 µg RNA second elution using same column could be performed
  24. Store @ -20 or -70 °C

RNA isolation for Real time qPCR: Saving the proteins

Extra material
  • 0.3 M guanidine hydrochloride (95.53 g/mol) in 95% EtOH
    • 0.28659 g / 10 mL 95% EtOH
Procedure
  1. Take the red, phenol/chloroform phase from step 7.1
  2. Precipitate DNA with 0.3 mL 100% EtOH per 1 mL TRIzol® Reagent
  3. Mix by inversion
  4. Incubate 2–3’ @ RT
  5. Centrifuge 15’ ≤2.000×g @ 4°C
  6. Collect supernatant (approx. 0.8 mL / mL TRIzol® Reagent)
    1. Pellet DNA can be used if desired for further processing (not included)
  7. Add isopropanol, 1.5 mL / mL TRIzol® Reagent
  8. Incubate 10’ @ RT
  9. Centrifuge 10’ 12.000×g @ 4°C
  10. Remove supernatant
  11. Add 2 mL 0.3 M guanidine hydrochloride in 95% EtOH / mL TRIzol® Reagent
  12. Incubate 20’ @ RT
  13. Centrifuge 5’ 7.500×g @ 4°C
  14. Remove supernatant
  15. Repeat step 35 – 38 up to a total of 3 times
  16. Add 2 mL of EtOH to the protein pellet
  17. Incubate 20’ @ RT
  18. Centrifuge 5’ 7.500×g @ 4°C
  19. Remove supernatant and dry pellet
  20. Resuspend pellet in 1% SDS solution (if necessary incubate @ 50°C to dissolve)
  21. Centrifuge 10’ 10.000×g @ 4°C
  22. Transfer supernatant to fresh tube
  23. Store @ -20°C

Attachment

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