Jacobs:Protocol Real-Time PCR

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)

Contact

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

or instead, discuss this protocol.