Jacobs:Protocol Real-Time PCR

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* RNase away
* RNase away
* Marker
* Marker
 +
 +
===Solutions===
 +
Reverse Transcription
 +
Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):
 +
 +
 +
For 1 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==
==Procedure==
-
# Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
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* Reverse Transcription
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## Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
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#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).
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##In tube #1, put in 120 µl of BSA, in all other tubes put in 60 µl of RIPA lysis Buffer (you always want to use the same dilutant material as what you used to isolate your protein)
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#Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.
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##Pipette 60 µl of BSA from tube #1 into #2.  Pipette up and down a dozen times or until you think it is properly mixed.  Then take 60 µl from tube #2 and put it in tube #3 and pipette up and down.  Continue this process through tube #5 (this will leave tube #5 with 120 µl)
+
#Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):
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#Prepare BCA Working Reagent
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*      20ºC 10 min
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##For the total volume of working reagent calculate:
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*37ºC 30 min
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##*(# standards (in our case 5) and samples (30 in our case))*(# replicates (2))*(volume of working solution per sample (200 µl))
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*99ºC 5 min
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##To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
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*4ºC 5 min (but set to 1 hr)
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#Prepare your samples
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##Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you  get within the range of 0.125-2 µl)
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##Freeze in -20ºC until further use.
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###Put 60 µl of your sample in tube S1
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###Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2
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* Real Time PCR
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###Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3
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# Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in triplicates): 
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#Prepare your Microplate
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## Taqman PCR Master Mix 2.5 μl
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##Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well
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## 20X Primer & Probe 0.25 μl x (N+1) Reactions
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##Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds
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## RNase free H2O 2 μl
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##Incubate plate at 37C  for 30 minutes
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Total Volume 4.75 μl
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##Remove plate and measure the absorbance at 562 nm on a plate reader
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##Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown
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#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.
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##Prepare a standard curve by plotting the average Blank=corrected 562 nm measurement for each BSA standard vs. its concentration in µg/µl.  Use the standard curve to determine the protein concentration of each unknown sample 
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#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.
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#Run in real time PCR machine.
 +
 
 +
 
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Real Time PCR Machine
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1. Centrifuge 384-well plate for 5 min at 2000 rpm.
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2. Use ABI PRISM 7900HT.
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3. Click SDS 2.1 icon.
 +
File – New – Absolute Quantification
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Add Detector – Select Genes and Click “Copy to Plate Document”
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In Setup pane, select regions of each gene and click “use”
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Task = unknown, Quantity = 0
 +
For standards,
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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==
==Notes==
-
Used at Stanford for Tissue Engineering Lab Course (ME385B and 2007 Winter/Summer TC Workshops
+
Used at Stanford for Tissue Engineering Lab Course (ME385B)
<!--
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#List troubleshooting tips here.   
#List troubleshooting tips here.   
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==Contact==
==Contact==
-
*Originally prepared by CRJ-EJC 1/3/06 
+
*Originally prepared by CRJ-EJC 3/1/04

Revision as of 21:17, 5 May 2008

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 1 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. 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)

References

Contact

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


or instead, discuss this protocol.

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