Jacobs:Protocol Real-Time PCR

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Contents

Overview

Protocol for Real-Time PCR

Materials

  • GeneAmp RNA PCR Core Kit (Part# N808-0143, Applied Biosystems)
  • PCR reaction tube
  • Centrifuge
  • 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
  • Marker

Solutions

Reverse Transcription

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

Procedure

  • Reverse Transcription
  1. 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).
  2. Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.
  3. 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)
  4. Freeze at -20ºC until further use.
  • Real Time PCR
  1. 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
  2. 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.
  3. Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.
  4. Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.
  5. 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.
  6. Add 0.25 μl of each dilution to wells corresponding to 18S Standards (5 different dilutions in triplicates; B1-B15).
  7. Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.
  8. Run in real time PCR machine.
  • Real Time PCR Machine
  1. Centrifuge 384-well plate for 5 min at 2000 rpm.
  2. Use ABI PRISM 7900HT.
  3. Click SDS 2.1 icon.
  4. File – New – Absolute Quantification
  5. Add Detector – Select Genes and Click “Copy to Plate Document”
  6. In Setup pane, select regions of each gene and click “use”
  7. Task = unknown, Quantity = 0
  8. For standards,Task = standard, Quantity = 16, 8, 4, 2, 1, Passive Ref = ROX
  9. In Instrument pane, Sample Volume = 13 l
  10. In Real Time pane, click “open/close” to open. Place plate, then click “close”.
  11. Save Changes – “*.sds”

Notes

Used at Stanford for Tissue Engineering Lab Course (ME385B)

References

Contact

  • Originally prepared by CRJ-EJC 3/1/04


or instead, discuss this protocol.

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