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 (from Step 1) 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)

1. 1. Freeze in -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 triplicates):
   1. Taqman PCR Master Mix 2.5 μl
   2. 20X Primer & Probe 0.25 μl x (N+1) Reactions
   3. RNase free H2O 2 μl

Total Volume 4.75 μl

1. 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.
2. Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.
3. Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.
4. 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).

1. Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.
2. 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. 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”

Notes

Used at Stanford for Tissue Engineering Lab Course (ME385B)

Contact

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

or instead, discuss this protocol.