qPCR set up and analysis (With Biorad reagents) - KravitzLab/KreedLabWiki GitHub Wiki

• PCR plate
• SYBR green
• USB stick
• P200 -P20 -P10
• Sterile tips -Ice -PCR machine (Here QuantStudio Pro 6)
• PCR plate lids
• Centrifuge
• cDNA
• RNAse free water

The first thing to do when making the PCR reagents is to determine how much you will need for your plate. A typical PCR plate is composed of 96 wells

Rows are numbered from A to H and column from 1 to 12. Typically, One row = One gene and 3 column = One individual. Ex : I wanna do the qPCR for animals A, B, C, D for genes 1, 2, 3, 4, 5, 6, 7, 8. This is what the PCR plate will look like

(You can put marker on the PCR plate and annotates it, I usually put the name of the gene on the left or right side and the subject name above the corresponding 3 column sections with a delimitation line).

So if you are doing 8 genes, you will use 3*8 wells with 3 being the number of replicates (here, 3) and 8 being the number of genes (here, 8). However, to not fall short and because of pipetting error, we will do all of our reagents for n+2 wells. So instead of making the reagents for 24 wells, you will make the reagent for 26 wells just to be sure that you will have enough to fill the PCR plate.

The amount of reagent is the following per well :

• 7.5 uL of SYBR green
• 4.5 uL of RNAse free water
• 0.5 uL of cDNA

For a total of 12 uL.

Therefore, for subject A you will make an eppendorf with 7.5 * 26 = 195 uL of SYBR green ; 4.526 = 117 uL of RNAse free water ; 0.526 = 13 uL of cDNA for the subject A.

/!\ MAKING THE REAGENT KEEP THE SYBR GREEN ON ICE

Make all your reagents for the 4 individuals.

That seem pretty easy but there is a specific way to fill the qPCR plate.

1- Once you have your reagent, centrifuge them for a few seconds to make sure all is mixed. DO NOT VORTEX !!!!!!

2- Take 12uL of the reagent for individual A gene 1 and put it in the first well (A1) by releasing the reagent on the LEFT SIDE OF THE WELL.

3- Using the same tips or different tips, put 12uL of the reagent for individual A gene 2 (A2) by releasing the reagent ON THE LEFT SIDE OF THE WELL

4- Continue to go down the line this way following the arrows ( )

Why proceed this way ? Because in case you are short in your reagent (pipetting mistake for example) and you miss reagent for 2 wells, instead of gene 8 having one replicate and 2 empty wells (NOT EXPLOITABLE), gene 7 and gene 8 will have 2 replicates instead of 3 (EXPLOITABLE).

Once you are done with filling up the plate with the reagents, it's time to add the primers

5- Take out the primers from the fridge

6- Wait for them to thaw at room temperature.

7- Gently vortex the primer solution you made (see page on primer)

8- Take 3uL of the primer for the gene A and put it in the first well (A1) on the RIGHT SIDE OF THE WELL.

The left/right side drop for the reagent and primer solution is to avoid contamination while also avoiding to change tips every time. Having the reagent and primer mix dropped on 2 opposite surface of the well make sure that the tip of the pipette containing the primers is not in contact with the mix with cDNA.

9- Continue to add the primers following the rows this way :

10- Once you are done with primers and your plate it filled, GENTLY tape it on the table so the drop reach the bottom of the well

11- Seal the plate with a plastic cover/lid. Make sure it is sealed properly (I use an old tips box to really push the plastic cover on the PCR plate)

12- Centrifuge the PCR plate for 30 seconds

And you are ready to load in your PCR and start the PCR !

1- Open the PCR software "Design and analysis" and click on "Standard Curve with melt"

2- in the "Run method" section, choose the qPCR parameters accordingly, IN THE PICTURE BELOW YOU HAVE THE SETUP USED FOR QPCR BIORAD EXCEPT THAT IT SAYS 20uL WHEN IT SHOULD BE 15 uL

3- In the plate setup set up the "virtual" PCR plate so that it matches your plate

SAMPLES : Name of the subject TARGET : Name of the gene.

PASSIVE REFERENCE FOR BIORAD SHOULD BE SET TO NONE

4- Once you are done, click on the Action and "Save as" to save your plate, its always useful to keep them to avoid remaking the same set up when you run different individuals for the same genes.

5- On "Run summary" click on the machine and click on "Send to Run queue"

6- On the PCR machine, click on the top right corner to load up the plate

7- Then tap on "Set up run", select the plate you just sent to the machine through the software

8 - Tap on "Start run".

9- Wait for about 1h30 minutes for the results

1- Once the PCR is done, export the data by plugging a USB stick to the machine and chose "Transfer file" and "USB stick".

2- Once it's transfered, plug the USB stick to the computer next to the PCR machine and open the file

3- Go on action and export and export the csv files to the USB stick

4- Remove the USB stick and plug it in your computer, open the file that contains "Results"

For all individuals, you should have 3 values for each gene because we run qPCR in triplicates. What matters ithe average value of the triplicates. In theory, the triplicates should be very close or nearly identitcal. Ex. For the gene 36B4 (Control gene for qPCR used in food intake analysis), individual A should have the following (MADE UP) values

Triplicate 1 : 24.2

Triplicate 2 : 24.1

Triplicate 3 : 24.5

Average = 24.23

In the case you have a triplicate that is one Cq value different from the other, discard it. Ex : still for 36B4

Triplicate 1 : 24.2

Triplicate 2 : 24.1

Triplicate 3 : 27

27 is to be excluded because it's higher than 24.2 and 24.1 by more than one cq value. So the average should be based on the 2 remaining values

Average = 24.15

Proceed to do that for all the genes and all individuals.

The analysis of the qPCR results is based on the 2(-Delta Delta C(T)) method : https://pubmed.ncbi.nlm.nih.gov/11846609/

Let's imagine this made up data set of average Cq value :

Cq	Control1	Control2	Control3	Exp1	Exp2	Exp3
36B4	24.1	24.3	23.5	23.8	24.6	24.5
Gene B	29.2	28.8	29.3	29.8	30.2	29.9

Control1, Control2, Control3 are 3 individuals of the control group. Exp1, Exp2, Exp3 are 3 individuals of the experimental group. 36B4 is the housekeeping or control gene, it's the gene that will serve to normalize the Cq value. Gene B is a gene of interest, we wanna see if it's more or less expressed in the experimental group.

The first thing to do is to substract 36B4 to Gene B for all individuals, it will give you the Delta Cq value

DCQ	Control1	Control2	Control3	Exp1	Exp2	Exp3
36B4	24.1	24.3	23.5	23.8	24.6	24.5
Gene B	5.1	4.5	5.8	6	5.6	5.4

Then you take the average of DCQ for Gene B in the control group ((5.1 + 4.5+5.8) / 3 = 5.13) And you substract this value to all values of Gene B, that will give you the Delta Delta Cq

DDCQ	Control1	Control2	Control3	Exp1	Exp2	Exp3
36B4	24.1	24.3	23.5	23.8	24.6	24.5
Gene B	-0.03333	-0.63333	0.666667	0.866667	0.466667	0.266667

Then to get the relative expression, you calculate the 2^(-DDCQ) value of the Gene B.

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ER	Control1	Control2	Control3	Exp1	Exp2	Exp3
36B4	24.1	24.3	23.5	23.8	24.6	24.5
Gene B	1.023374	1.551145	0.629961	0.548412	0.723635	0.831238

This will give you the relative expression of your gene compared to the control group. Therefore, the control group should have values close to 1 and and average of about 1, which is the case here ((1.02 + 1.55 + 0.63) / 3) = 1.06

And for the experimental group, if the value is below 1 its a lower expression, if it's higher than 1, it's a higher expression

Here the average is 0.70 for the experimental group. So we can see here that the expression is lower in the experimental group (Of course, you'll need to confirm with stats).

My Cq value are all over the place

Several things can cause this issue

• Pipetting mistake is the most common cause. Make sure to be extra careful when pipetting in the wells
• You didn't thaw the primer mix. That can make the Cq value look weird
• You have contamination in your PCR : That's usually mean that your primer mix is contaminated by something. Look at the melting curve, it should have one peak for all individuals located around the same temp

On the left : Normal melting curve with one peak.
On the right : Melting curve with 2 peaks. The first peak is likely a contamination OR an off-target amplification.

If your primer set are known to be specific and you didn't have this issue before, it's likely that it's contamination. The easiest solution is to remake the primer mix !

The relative expression of my control group is not equal to 1

So admitting everything is well with the qPCR and Cq value are not all over the place, the primers are specific, the curves look fine, then it comes down to 2 potential issues.

• You do not have enough individuals in your control group. Usually it's good to aim for 8-12 individuals to make sure to have an average close to 1.
• The expression of some genes is sometimes all over the place even in control condition.

_Ex : _

NAPE (N-acylphosphatidilethanolamine, involved in endocannabinoid synthesis) expression in the VTA in rats is highly variable even in control conditions _(Schoukroun et al., 2025) _

. In that case there isn't a good solution (increasing the effective can help or worsen the variability), its possible that if there is a difference between the gene expression between the two groups you would still be able to see unless the difference is rather small.