Jacobs:Protocol Real-Time PCR

Overview
Protocol for RT 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):
 * 4) *20ºC 	10 min
 * 5) *37ºC	30 min
 * 6) *99ºC	5 min
 * 7) *4ºC	5 min (but set to 1 hr)
 * 8) 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):
 * 2) * Taqman PCR Master Mix 		2.5 μl
 * 3) * 20X Primer & Probe			0.25 μl		x (N+1) Reactions
 * 4) * RNase free H2O			2 μl
 * 5) *Total Volume		4.75 μl
 * 6) 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.
 * 7) Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.
 * 8) Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.
 * 9) 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.
 * 10) Add 0.25 μl of each dilution to wells corresponding to 18S Standards (5 different dilutions in triplicates; B1-B15).
 * 11) Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.
 * 12) 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”

Contact

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

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