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real_time_pcr [2011/01/19 13:23]
csbq_qcbs 		real_time_pcr [2012/04/16 12:25]
anniearchambault
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 The real-time PCR experiment takes place in a thermocycler equipped with a camera to read fluorescence emission. ​ The real-time PCR experiment takes place in a thermocycler equipped with a camera to read fluorescence emission. ​
  
-The Wikipedia page [[wp>en.wikipedia.org/​wiki/​Real_time_PCR]] is useful for a more elaborate background +The Wikipedia page [[wp>​Real_time_PCR]] is useful for a more elaborate background. The Agilent guide [[http://​www.genomics.agilent.com/​campaigns/​qpcrguide/​|Introduction to Quantitative PCR: Methods and Applications Guide]] is also a very informative resource. This is an augmented version of the  {{:​stratagene_introduction_to_quantitative_pcr_methods_and_application_guide.pdf|Stratagene Introduction to Quantitative PCR 
 +Methods and Application Guide}}
  
 ===Different real time PCR methods=== ===Different real time PCR methods===
-Using double stranded DNA binding dyes, SYBR green I or SYBR green I for instance, to detect the amount of amplified DNA in real time is the most commonly used method in biodiversity science. Main advantages are that it is easy to set up and convenient, since the user has only to optimize forward and reverse primers sequences and reaction conditions. Limitations are that the double stranded DNA binding dyes will also anneal to nonspecific amplified products and primers dimers. ​+Using double stranded DNA binding dyes, SYBR green I or SYBR green II for instance, to detect the amount of amplified DNA in real time is the most commonly used method in biodiversity science. Main advantages are that it is easy to set up and convenient, since the user has only to optimize forward and reverse primers sequences and reaction conditions. Limitations are that the double stranded DNA binding dyes will also anneal to nonspecific amplified products and primers dimers. ​
  
-Sequence specific fluorescent probes (TaqMan probes) anneal to the target DNA fragment and will emit their fluorescence when degraded by the 5' to 3' exonuclease activity of the polymerase. The Wikipedia page [[wp>http://​en.wikipedia.org/​wiki/​TaqMan]] explains in more detail the process. This method has the advantage of increased specificity,​ since the probe will not anneal to nonspecific amplified products and primers-dimers. It also enables the multiplexing of up to four or five reactions in a single tube, when using probes attached to different fluorescent molecules. The method is however more complex to set up, as the user will need to optimize the sequence and reaction conditions of the specific probe in combination with specific amplification primers. Moreover, because the probe is sequence specific, it cannot be transferred to another experiment. ​+Sequence specific fluorescent probes (TaqMan probes) anneal to the target DNA fragment and will emit their fluorescence when degraded by the 5' to 3' exonuclease activity of the polymerase. The Wikipedia page [[wp>​TaqMan]] explains in more detail the process. This method has the advantage of increased specificity,​ since the probe will not anneal to nonspecific amplified products and primers-dimers. It also enables the multiplexing of up to four or five reactions in a single tube, when using probes attached to different fluorescent molecules. The method is however more complex to set up, as the user will need to optimize the sequence and reaction conditions of the specific probe in combination with specific amplification primers. Moreover, because the probe is sequence specific, it cannot be transferred to another experiment. ​
  
- 
- 
-===Performing Real time PCR at the QCBS=== 
-There is a MxPro3005 Stratagene instrument in Daniel Schoen lab from biology department at McGill, which has the following characteristics. Everyone who wish to use the instrument should make arrangement with [[http://​biology.mcgill.ca/​faculty/​schoen/​|Daniel Schoen]]. It should also be noted that training for using the instrument cannot be provided by members of the Schoen lab.    
- 
-Annie Archambault (QCBS research professional) and Abderrahman Khila (from Ehab Abouheif lab) have been setting up that instrument in December 2010. The experiments were sat up using SYBR green I.  
  
  
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 A minimal of two technical replicates need to be analysed, and three biological replicates are routinely included for each sample.  ​ A minimal of two technical replicates need to be analysed, and three biological replicates are routinely included for each sample.  ​
  
-===A typical ​plate setup for a real time qPCR with SYBR green dye===+===A typical ​reaction mix for a real time qPCR with SYBR green dye===
 {{  :​platesetup_qpcr_mxpro.jpg?​250|Typical plate setup for real time PCR on a MxPro3005}} {{  :​platesetup_qpcr_mxpro.jpg?​250|Typical plate setup for real time PCR on a MxPro3005}}
-We used 15 ul reaction, instead of 25 ul or 50 ul reactions. Experiments were done using 8-strips tubes. ​+At the Abouheif lab, we used 10 ul volume ​reaction, instead of 25 ul or 50 ul reactions. Experiments were done using 8-strips tubes. ​Although the one should follow the manufacturer protocol, we list here the ingredients for reaction mix, for information purposes only.  ​
  
-Each tube contains +Each tube contained 
-  *n ul of SYBR green mix (for instance Agilent Brilliant II SYBR Green QPCR master mix) +  *5 ul of SYBR green mix (for instance Agilent Brilliant II SYBR Green QPCR master mix; or BioRad SYBR Green Supermix cat. no. 170-8880) 
-  *n ul of Formard Primer at n uM +  *1 ul of Formard Primer at 2 uM 
-  *n ul of Reverse Primer at n uM +  *1 ul of Reverse Primer at 2 uM 
-  *n ul of cDNA, (diluted 1/50 relative to original tube) +  *1 ul of cDNA (diluted 1/50 relative to original tube) 
-  *n ul of ROX  +  *1 ul of ROX (diluted 1/250) 
-  *Water+  *2 ul of Water
  
  
-An experiment where the expression ratio for five genes is compared in four plant organs under two growth conditions may require the set up of up to 680 reactions (wells).  ​+ 
 +===A typical plate set up for a real time qPCR with SYBR green dye=== 
 +An hypothetical ​experiment where the expression ratio for five genes is compared in four plant organs under two growth conditions may require the set up of up to 680 reactions (wells).  ​
   * 5 genes: MN, CB, RP, HF, SL   * 5 genes: MN, CB, RP, HF, SL
   * 1 calibrator gene: tubulin   * 1 calibrator gene: tubulin
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 **Figure 1. Plate setup for a qPCR experiment, on one gene (MN), one condition (High CO2), four organs (total of 68 wells)** **Figure 1. Plate setup for a qPCR experiment, on one gene (MN), one condition (High CO2), four organs (total of 68 wells)**
-|  ^ 1  ^ 2  ^ 3  ^ 4  ^ 5  ^ 6  ^ 7  ^ 8  ^ 9  ^ 10  ^ 11  ^ 12  ^  +| ^ 1 ^ 2 ^ 3 ^ 4 ^ 5 ^ 6 ^ 7 ^ 8 ^ 9 ^ 10 ^ 11 ^ 12 ^  
-^ A | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ A  | Standard; Calibrator 10 E-4 | Standard; Calibrator 10 E-8 | Unknown; Calibrator cDNA High CO2, shoot, br1 | Unknown; Calibrator cDNA High CO2, leaves, br2 | Unknown; Calibrator cDNA High CO2, fp, br3 | Standard; Gene MN 10 E-7 | Unknown; Gene MN cDNA High CO2, root, br3 | Unknown; Gene MN cDNA High CO2, leaves, br1 | Unknown; Gene MN cDNA High CO2, fp, br2 | empty well | empty well | empty well |  
-^ B | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ B  | Standard; Calibrator 10 E-4 | Standard; Calibrator 10 E-8 | Unknown; Calibrator cDNA High CO2, shoot, br1 | Unknown; Calibrator cDNA High CO2, leaves, br2 | Unknown; Calibrator cDNA High CO2, fp, br3 | Standard; Gene MN 10 E-7 | Unknown; Gene MN cDNA High CO2, root, br3 | Unknown; Gene MN cDNA High CO2, leaves, br1 | Unknown; Gene MN cDNA High CO2, fp, br2 | empty well | empty well | empty well |  
-^ D | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ C | Standard; Calibrator 10 E-5 | Unknown; Calibrator cDNA High CO2, root, br1 | Unknown; Calibrator cDNA High CO2, shoot, br2 | Unknown; Calibrator cDNA High CO2, leaves, br3 | Standard; Gene MN 10 E-4 | Standard; Gene MN 10 E-8 | Unknown; Gene MN cDNA High CO2, shoot, br1 | Unknown; Gene MN cDNA High CO2, leaves, br2 | Unknown; Gene MN cDNA High CO2, fp, br3 | empty well | empty well | empty well |  
-^ E | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ D  | Standard; Calibrator 10 E-5 | Unknown; Calibrator cDNA High CO2, root, br1 | Unknown; Calibrator cDNA High CO2, shoot, br2 | Unknown; Calibrator cDNA High CO2, leaves, br3 | Standard; Gene MN 10 E-4 | Standard; Gene MN 10 E-8 | Unknown; Gene MN cDNA High CO2, shoot, br1 | Unknown; Gene MN cDNA High CO2, leaves, br2 | Unknown; Gene MN cDNA High CO2, fp, br3 | empty well | empty well | empty well |  
-^ F | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ E  | Standard; Calibrator 10 E-6 | Unknown; Calibrator cDNA High CO2, root, br2 | Unknown; Calibrator cDNA High CO2, shoot, br3 | Unknown; Calibrator cDNA High CO2, fp, br1 | Standard; Gene MN 10 E-5 | Unknown; Gene MN cDNA High CO2, root, br1 | Unknown; Gene MN cDNA High CO2, shoot, br2 | Unknown; Gene MN cDNA High CO2, leaves, br3 | Non template containing Calibrator gene | empty well | empty well | empty well |  
-^ G | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ F  | Standard; Calibrator 10 E-6 | Unknown; Calibrator cDNA High CO2, root, br2 | Unknown; Calibrator cDNA High CO2, shoot, br3 | Unknown; Calibrator cDNA High CO2, fp, br1 | Standard; Gene MN 10 E-5 | Unknown; Gene MN cDNA High CO2, root, br1 | Unknown; Gene MN cDNA High CO2, shoot, br2 | Unknown; Gene MN cDNA High CO2, leaves, br3 | Non template containing Calibrator gene  | empty well | empty well | empty well |  
-^ H | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 | Row 1 Col 2 | Row 1 Col 3 |  +^ G  | Standard; Calibrator 10 E-7 | Unknown; Calibrator cDNA High CO2, root, br3 | Unknown; Calibrator cDNA High CO2, leaves, br1 | Unknown; Calibrator cDNA High CO2, fp, br2 | Standard; Gene MN 10 E-6 | Unknown; Gene MN cDNA High CO2, root, br2 | Unknown; Gene MN cDNA High CO2, shoot, br3 | Unknown; Gene MN cDNA High CO2, fp, br1 | Non template containing MN gene | empty well | empty well | empty well |  
 +^ H  | Standard; Calibrator 10 E-7 | Unknown; Calibrator cDNA High CO2, root, br3 | Unknown; Calibrator cDNA High CO2, leaves, br1 | Unknown; Calibrator cDNA High CO2, fp, br2 | Standard; Gene MN 10 E-6 | Unknown; Gene MN cDNA High CO2, root, br2 | Unknown; Gene MN cDNA High CO2, shoot, br3 | Unknown; Gene MN cDNA High CO2, fp, br1 | Non template containing MN gene | empty well | empty well | empty well | 
  
 This plate setup would need to be done for each of the five genes, and each of the two conditions. ​ This plate setup would need to be done for each of the five genes, and each of the two conditions. ​
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   * Agilent Mx3000P/​Mx3005P Optical Strip Caps and tubes (cat no 401428 ​ and 401425): 0.17$ per well   * Agilent Mx3000P/​Mx3005P Optical Strip Caps and tubes (cat no 401428 ​ and 401425): 0.17$ per well
  
-The example given above, for 24 biological samples and 680 real time reaction, may cost approximately a thousand dollars in consumable plastic and reagents.+The example given above, for 24 biological samples and 680 real time reactions, may cost approximately a thousand dollars in consumable plastic and reagents. ​ 
 + 
 + 
 +====Performing Real time PCR at the QCBS==== 
 +There is a MxPro3005 Stratagene instrument in Daniel Schoen lab from biology department at McGill, which has the following characteristics. Everyone who wish to use the instrument should make arrangement with [[http://​biology.mcgill.ca/​faculty/​schoen/​|Daniel Schoen]]. It should also be noted that training for using the instrument cannot be provided by members of the Schoen lab.    
 + 
 +Annie Archambault (QCBS research professional) and Abderrahman Khila (from Ehab Abouheif lab) have been setting up that instrument in December 2010. The experiments used SYBR green I, but Brilliant SYBR green II is now most widely used.