Jacobs:Protocol RNA Isolation Using Tri Reagent
Protocol for Real-Time PCR
Tri-Reagent (Sigma T9424) Cell scraper Pipetman Pipet tips Autoclaved microcentrifuge tube Chloroform (Sigma (C2432) Centrifuge Isopropanol (Sigma I9516) 75% Ethanol Kimwipe RNase free H2O Spectrophotometer
1. Lyse cells directly on culture dish. Add 1 ml of TRI REAGENT (for up to 5x106 cells). Use cell scraper to scrape off cells and pass cell lysate through pipette 20 times to form homogeneous lysate. (Perform Steps 1-2 in fume hood)
- Note: If using plastic dish, work as quickly as possible since TRI REAGENT is not compatible with plastic.
2. Transfer lysate to 1.5 ml microcentrifuge tube. 3. Allow samples to stand for 7 mins at room temperature for complete dissociation of nucleoprotein complexes. 4. Add 0.2 ml of chloroform. Cover the sample, vortex for 10 secs and allow to stand for 10 mins at room temperature. Centrifuge the resulting mixture at 12,000g for 15 mins at 4C. This separates the mixture into 3 phases: a red organic phase (protein) in the bottom, an interphase pellet (DNA) in the middle, and a colorless aqueous phase (RNA) at the top.
RNA Isolation 1. Transfer top aqueous phase to a new autoclaved microcentrifuge tube and add 0.5 ml of isopropanol. Mix well and allow sample to stand for 10 mins at room temperature.
- Note: Store the interphase and organic phase at 4C for subsequent isolation of DNA and proteins.
2. Centrifuge at 12,000g for 10 mins at 4C. The RNA precipitate will form a pellet on the bottom of the tube. 3. Remove the supernatant and wash RNA pellet by filling tube with 75% ethanol (~1.5 ml). Vortex sample and then centrifuge at 7,500g for 5 mins at 4C.
- Note: Samples can be stored in ethanol at 4C for 1 wk and at -20C for 1 year.
4. Pour out ethanol (be careful not to pour out RNA pellet). Place tubes upside down on Kimwipe to rid of trace of ethanol. Do not let the RNA pellet dry completely. 5. Add 20 l RNase free H2O to RNA pellet. Place tubes on ice. Resuspend pellet and transfer 1 l into new tube with 99 l RNase free H2O for RNA quantification. 6. Turn on spectrophotometer (switch is on back of machine) let it self calibrate (~3 mins) 7. Turn on UV lamp (takes ~ 10 mins): Function – Setup – Lamps – UV and Visible (325 nm) 8. Wash cuvette with H2O. 9. Mode – Nucleic – RNA – Background ON. Then reference with 100 l H2O. 10. Remove H2O. 11. Transfer sample in cuvette (100 l from Step 5) and run. 12. Write down ratio and absorbance at 260, 280.
- Note: Concentration (g/ml) = [(A260-A280)x100x40]
Total Yield (g) = Conc x 0.019 260 to 280 ratio should be greater than 1.7. 13. Turn off power. 14. Store RNA at -20C.
- Reverse Transcription
- In PCR reaction tube, add 20 μL of Master Mix (above) and 20 μL of RNA sample (appropriate amount containing 3 μg of RNA and then q.s. to 20 μL). Centrifuge briefly (~10 sec).
- Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.
- Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):
- 20ºC 10 min
- 37ºC 30 min
- 99ºC 5 min
- 4ºC 5 min (but set to 1 hr)
- Freeze at -20ºC until further use.
- Real Time PCR
- Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in triplicate):
- Taqman PCR Master Mix 2.5 μl
- 20X Primer & Probe 0.25 μl x (N+1) Reactions
- RNase free H2O 2 μl
- Total Volume 4.75 μl
- Add 4.75 μl of mixed reagents to each well corresponding to specific genes (in this lab, we are only looking at 18S; the wells are A1-A12 and B1-B15) in the 384-well plate.
- Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.
- Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.
- For 18S (house keeping gene) standards, prepare five 1.5 ml microcentrifuge tubes and label them 1, 2, 3, 4, 5. Add 9 μl RNase free H2O in all five tubes. Add 1 μl of cDNA (use any one of Sample cDNA) in #1. Mix well and transfer 1 μl from tube 1 to tube 2 (10-fold dilution). Mix well and transfer 1 μl from tube 2 to tube 3. Mix well and transfer 1 μl from tube 3 to tube 4. Mix well and transfer 1 μl from tube 4 to tube 5.
- Add 0.25 μl of each dilution to wells corresponding to 18S Standards (5 different dilutions in triplicates; B1-B15).
- Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.
- Run in real time PCR machine.
- Real Time PCR Machine
- Centrifuge 384-well plate for 5 min at 2000 rpm.
- Use ABI PRISM 7900HT.
- Click SDS 2.1 icon.
- File – New – Absolute Quantification
- Add Detector – Select Genes and Click “Copy to Plate Document”
- In Setup pane, select regions of each gene and click “use”
- Task = unknown, Quantity = 0
- For standards,Task = standard, Quantity = 16, 8, 4, 2, 1, Passive Ref = ROX
- In Instrument pane, Sample Volume = 13 l
- In Real Time pane, click “open/close” to open. Place plate, then click “close”.
- Save Changes – “*.sds”
Used at Stanford for Tissue Engineering Lab Course (ME385B)
- Originally prepared by CRJ-EJC 2/17/04
or instead, discuss this protocol.