RNA isolation from cells | RNA quantification

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