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real_time_pcr

Quantitative real time PCR

q-real-time PCR is a laboratory technique where the amplified DNA is detected at each cycle, as the reaction progresses in real time. Two common methods for detection of DNA amplified products in real-time PCR are:

  • Using double stranded DNA binding dyes (mainly SYBR green)
  • Using short sequence specific fluorescent DNA probes (TaqMan probes)

The real-time PCR experiment takes place in a thermocycler equipped with a camera to read fluorescence emission.

The Wikipedia page Real_time_PCR is useful for a more elaborate background. The Agilent guide 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

Different real time PCR methods

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 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.

Required reagents and plastics for a real time qPCR with SYBR green dye experiment

  • SYBR green mix the mixes usually contain the polymerase, nucleotides, salts, sufficient MgCl2, SYBR green I or SYBR green II as DNA-binding fluorescent dyes. The mixes may also contain a reference dye such as Fluorescein or ROX, that does not bind to DNA or interfere with amplification, but that will be used for internal calibration by the instrument.
  • Primers Primers should have very similar annealing temperature, and should ideally amplify a region of 100 to 200 bp. It is wise to take into account the intron location. A primer can be designed to encompass an intron location to prevent amplification from genomic DNA, or the primers can be designed in two different exons to enable the detection of genomic DNA amplification, that show as a longer amplified fragment than the cDNA.
  • cDNA A pure cDNA exempt of genomic DNA, and PCR inhibitors, made from non-degraded RNA is critical. If the cDNA is highly concentrated, it is suggested to dilute it in 1/20 or 1/50 in order to save precious cDNA.
  • Optical tubes or plates tubes or plates used must be compatible with fluorescence detection. Consult manufacturer catalog to confirm that the plates and tubes can be used with your instrument.
  • A set of repeater pipettes may come useful.

Calibrators

Real-time PCR can be used to quantify nucleic acids by two strategies: Absolute quantification and Relative quantification. Relative quantification measures the fold-difference (2X, 3X etc.) in the amount of the target gene compared to the expression of a calibrator or normalizer gene that keeps a constant expression level across conditions, tissue and taxa. Absolute quantification gives the exact number of target molecules present by comparing with known standards. Relative quantification is the most commonly used method in biodiversity science.

When performing an real-time experiment using relative quantification, the user will therefore have to design primers, not only to the genes under study, but also to the calibrator gene (or to more than one calibrator gene) for the species.

Standards

In downstream calculations of expression levels from fluorescence levels, the efficiency of the PCR amplification for a given set of primers will be used to adjust the expression levels. Standards are done by preparing serial dilutions of a purified PCR product, and using at least five dilutions as templates for a real-time PCR experiment.

Duplicates

A minimal of two technical replicates need to be analysed, and three biological replicates are routinely included for each sample.

A typical reaction mix for a real time qPCR with SYBR green dye

Typical plate setup for real time PCR on a MxPro3005 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 contained

  • 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)
  • 1 ul of Formard Primer at 2 uM
  • 1 ul of Reverse Primer at 2 uM
  • 1 ul of cDNA (diluted 1/50 relative to original tube)
  • 1 ul of ROX (diluted 1/250)
  • 2 ul of Water

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
  • 1 calibrator gene: tubulin
  • 4 organs: root; shoot; leaves, floral primordium (fp)
  • 2 conditions: normal CO2; high CO2
  • 3 biological replicates
  • 2 technical replicates

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
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 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
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
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
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
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
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.

Estimation of costs

The main cost will come from :

  • Qiagen RNeasy Plant Mini Kit RNA extraction kits (cat no 74904): at least 5.05$ per biological sample
  • Qiagen QuantiTect Reverse Transcription Kit (cat no 205311): 7.30$ per biological sample
  • Agilent Brilliant II SYBR Green QPCR master (cat no 600828): 0.65$ per reaction (for 15 ul volume reactions)
  • 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 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 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.

real_time_pcr.txt · Last modified: 2012/04/16 12:25 by anniearchambault