Jacobs:Protocol Real-Time PCR
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Protocol for RT PCR
- GeneAmp RNA PCR Core Kit (Part# N808-0143, Applied Biosystems)
- PCR reaction tube
- Liquid Wax
- Thermal cycler
- RNase free Microcentrifuge tubes
- RNase free H2O
- Taqman PCR Master Mix (Applied Biosystems 4304437)
- 20X Primers and Probes (Applied Biosystems)
- 384-well plate (Applied Biosystems 4309849)
- Optical cover (Applied Biosystems 4311971)
- Light mineral oil (Fisher M5904)
- Real Time PCR machine
- Various pipet tips and pipetter
- RNase away
Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):
For one reaction
- MgCl2 8 μL
- 10X PCR Buffer 4 μL
- dGTP 0.4 μL x (N+1) for N reactions
- dATP 0.4 μL x (N+1) for N reactions
- dTTP 0.4 μL x (N+1) for N reactions
- dCTP 0.4 μL x (N+1) for N reactions
- RNase Inhibitor 2 μL
- Reverse Transcriptase 2 μL
- Random Hexamer 2 μL
- DEPC Treated H2O 0.4 μL
- 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 3/1/04
or instead, discuss this protocol.