RNA isolation from cells | RNA quantification

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RNA isolation from cells

Protocol:


  • Aspirate off media, wash once with ice cold PBS (1-2) mL.
  • Aspirate off PBS, add 1 mL Trizol.
  • Scrape the plate with a cell scraper, collect the cell lysate to 1.5 mL E-tube.
  • Incubate RT for 5mins.
  • Centrifugation 4°C, 13000 rpm for 10 mins.
  • Aliquot supernatant to new E-tube.
  • Add 200 µL chloroform (200 µL + 800 µL=1 mL), vortexing.
  • Incubate RT for 10-15 mins (vortex every 2 mins)
  • Centrifugation 4°C, 13000 rpm for 15 mins.
  • Separate aqueous phase and transfer to new E-tube.
  • Add isopropanol (equal volume of aqueous phase)
  • Inverting, incubate RT for 5-10 mins.
  • Centrifugation 4°C, 13000 rpm for 10 mins.
  • Remove supernatant and add 700 µL 70%EtOH to wash the pellet.
  • Centrifugation 4°C, 13000 rpm for 5 mins.
  • Remove supernatant and add 1 mL 70%EtOH to wash the pellet.
  • Centrifugation 4°C, 7000 rpm for 10 mins.
  • Remove supernatant, spin down.
  • Use yellow tip remove supernatant completely.
  • Air dry rapping with poly for 5-10 mins.
  • Add DEPC-DW 35 µL to E-tube.
  • Spin down, qty/store -80°C.
Fig: Illustration of RNA isolation from cells


RNA quantification

Instruments:

  • 20 µL pipet
  • 100 µL pipet
  • Yellow & white tip
  • Tip discard box
  • Kim tissue
  • 70% EtOH
  • 75 µL tube & levelling
  • RNA sample
  • DEPC-DW
Fig: Denovix DS-11 Spectrophotometer from DeNovix Inc.


Protocol:

  • Place DEPC-DW & RNA sample on ice.
  • Turn on NANODROP machine (Location: 702)
  • Open and enter RNA button
  • Wipe the microplate using 70% EtOH
  • Add, 2 µL DEPC-DW to the microplate, press the measure button, then wipe the plate with 70% EtOH.
  • Touch the BLANK, wipe.
  • Add, 2 µL RNA sample to the microplate, press the measure button, then wipe the plate with 70% EtOH.
  • Store and transfer data to USB.
  • Calculate the vol. Of RNA samples for reverse transcription in excel.

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

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