Reaction mixes and qPCR

Now, let’s proceed to the qPCR.

Material

  • A real-time PCR machine (here : Applied BioSystems QuantStudio 5).
  • 1X 384-well plate (the same for all of you).
  • 5 µM Forward primers for all the pairs that you will test.
  • 5 µM Reverse primers for all the pairs that you will test.
  • qPCR master mix 2X (contains the Taq polymerase + dNTPs + buffer).
  • Sterile water.
  • PCR tubes.

Note: When performing the following steps, only use filtered tips and wear gloves!

OVERVIEW - Make sure you understand the information below before proceeding to the experiment. The procedure will be performed in successive steps :

The first step will be to produce a “Master mix” for each primer pair (= each gene) that is analyzed. These three mixes will all contain everything required for the qPCR, EXCEPT the cDNAs. The second step will be to divide this mix into three tubes : one for the amplification of the gene in the control sample, another for the amplification of the gene in the tested sample, and the last one will be used as a blank control. The third step will be to add the correct sample to these tubes : - 1. the control cDNA ; - 2. The mutant’s cDNA ; - 3. Water in the control tube. Finally, these mixes will be used to load the PCR.

Procedures

  1. First, prepare and annotate three 1,5 ml Eppendorf tubes. Each of these tubes will contain a specific qPCR mix, corresponding to any of the 3 genes (two reference genes + one candidate gene) that you will analyze. The tubes will thus be:

    • Tube 1 –> qCPR mix for Gene 1
    • Tube 2 –> qCPR mix for Gene 2
    • Tube 3 –> qCPR mix for Gene 3

  2. For each of these tubes, the mix consists in (DO NOT ADD THE cDNA at this step):

    • 48 µl Master Mix 2X
    • 3 µl primer FOR 5 µM GENE X (final concentration = 153 nM)
    • 3 µl primer REV 5 µM GENE X (final concentration = 153 nM)
    • 26 µl H2O.

  3. Slightly vortex and centrifuge the mix.

  4. Annotate the PCR tubes in which the mixes will be spread. Let’s use tube 1 as an example :

    • Tube 1A = Gene 1 (=primer pair 1), WT sample
    • Tube 1B = Gene 1 (=primer pair 1), mutant sample
    • Tube 1C = Gene 1 (=primer pair 1), qPCR negative control.

  5. Add the following volumes of the mix from Tube 1 to the tubes:

    • Tube 1A = 30 µl
    • Tube 1B = 30 µl
    • Tube 1C = 12 µl (we do not perform replicates for the negative control) qCPR protocol

  6. Proceed in the same way for the mixes 2 and 3. You should thus have 9 tubes.

  7. Now, we have to add the cDNAs to our samples:

    • Tube 1A,2A,3A = add 6 µl of cDNA from the WT sample
    • Tube 1B,2B,3B = add 6 µl of cDNA from the mutant sample
    • Tube 1C,2C,3C = add 3 µl of sterile water.

  8. Vortex and centrifuge the mixes.

  9. Your mixes are ready to be loaded in the 384-well plate: Load all your samples on one row (=1 ligne/groupe). Write on the page near the plate which row you used and which genes correspond to tubes 1, 2, and 3. THIS IS VERY IMPORTANT, AS WE NEED TO BE ABLE TO KNOW WHICH SAMPLE WAS LOADED AT WHICH POSITION IN ORDER TO LAUNCH THE qPCR. Any change in the sample order should also be noted here, but try to keep it to a minimum.

  10. Add 10 µl of the mix, the triplicates following each other :

    • 10 µl of 1A will be added to wells 1, 2, and 3 ; 10 µl of 1B to wells 4, 5, 6; 10 µl of 1C to well 7 (only one replicate).
    • 10 µl of 2A will be added to wells 8, 9, and 10 ; 10 µl of 2B to wells 11, 12, 13; 10 µl of 1C to well 14 (only one replicate).
    • 10 µl of 3A will be added to wells 15, 16, and 17 ; 10 µl of 3B to wells 18, 19, 20; 10 µl of 3C to well 21 (only one replicate). qCPR protocol

  11. After all groups have loaded their mixes, the plate will be covered with a transparent plastic to avoid any evaporation during the qPCR process.

  12. The last step is to launch the qPCR using the following program :

    1. 2 minutes at 50 °C
    2. 3 minutes at 95 °C
    3. 10 seconds at 95 °C
    4. 20 seconds at 57 °C
    5. 30 seconds at 72 °C -> Fluorescence measurement : back 39x to step 3
    6. After 40 cycles: final elongation of 7 minutes at 72 °C.

  13. After the end of the amplification, the machine will perform an extra step, which will help determine whether the amplification we performed was specific or not. For this purpose, it will perform the following steps :

    1. Initial denaturation : 1 minute at 95 °C
    2. Renaturation as double-stranded DNA : 1 minute at 60 °C
    3. Incremental increase of the temperature, from 60°C to 95°C (1°C/13,3 sec) –> Fluorescence measurement.

qCPR protocol

Note : to know to which gene of interest your primers correspond, you can refer to the image below:

qCPR protocol