Real Time Primer Efficiencies
For Use with SYBR Green
1. Before beginning real time experiments with a new set of primers, we suggest
doing a primer efficiency test. This is done to insure that the primers will work
appropriately and also to determine what concentration of cDNA to use in the
experiment. (This is only needed for SYBR Green experiments).
2. To determine the efficiency, you will need an internal control (housekeeping
gene) and your primer of interest. Set up reactions using a series of
concentrations of cDNA (typically 5). See the example below. (HKG = house
keeping gene) (POI = primer of interest)
A
B
C
D
E
1
HKG
1ng
HKG
1ng
POI
1ng
POI
1ng
RTHKG
5ng
2
HKG
2ng
HKG
2ng
POI
2ng
POI
2ng
RTPOI
5ng
3
HKG
3ng
HKG
3ng
POI
3ng
POI
3ng
NTC
HKG
4
HKG
4ng
HKG
4ng
POI
4ng
POI
4ng
NTC
POI
5
6
HKG
5ng
HKG
5ng
POI
5ng
POI
5ng
7
8
9
10
11
12
F
G
H
Always do your RT+ reactions in duplicate. RT- reactions always need to be run on a
plate with the RT+ reactions. You don’t have to do one for every amount of cDNA, but
instead can set up a RT- reaction with the highest amount of cDNA used on the plate.
RT- reactions can help you determine if you have primer dimers or DNA contamination.
NTC (no template control) reactions also need to be run on every plate to determine if
there is any DNA contamination.
3. Once you get the results, you need to do some calculations. First, you want to get
the mean and standard error of the duplicates. Below is an example of what you
might see for results and how to do the calculations.
Well
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
C1
C2
C3
C4
C5
D1
D2
D3
D4
D5
E1
E2
E3
E4
Sample
HKG 1ng
HKG 2ng
HKG 3ng
HKG 4ng
HKG 5ng
HKG 1ng
HKG 2ng
HKG 3ng
HKG 4ng
HKG 5ng
POI 1ng
POI 2ng
POI 3ng
POI 4ng
POI 5ng
POI 1ng
POI 2ng
POI 3ng
POI 4ng
POI 5ng
RT- HKG 5ng
RT- POI 5ng
NTC HKG
NTC POI
Ct
18.12
17.82
17.39
17.12
17.12
18.1
17.83
17.54
17.4
17.12
18.01
17.37
17.19
16.97
16.75
18.12
17.61
17.17
17.05
16.89
29.24
28.52
Undetermined
Undetermined
** Always make sure that your RT- reaction Ct value is at least 10 Ct values away from
your corresponding RT+ value. Undetermined means that the machine did not detect
fluorescence within the 40 cycles, which is what you expect for the no template control.
A. To get the mean and standard error of the mean between the replicates, we use
Graph Pad Prism 4.0. When you open the software, select “Create a New
Project” and “Format of data table.” For the X column, you want to select
“None (column graph).” You can leave the Y column at default.
B. Once your project is open, enter the Ct values by putting the duplicates in the
same column.
1
2
3
4
A
Title
Y
18.10
18.12
A
Title
Y
17.82
17.83
A
Title
Y
17.39
17.54
A
Title
Y
17.12
17.40
A
Title
Y
17.12
17.12
A
Title
Y
18.01
18.12
A
Title
Y
17.37
17.61
A
Title
Y
17.17
17.19
A
A
Title Title
Y
Y
16.97 16.75
17.05 16.89
C. After you have entered the values, select “Insert/NewAnalysis/Statistical
Analysis/Column Statistics/Create.” This analysis will give you the mean and
the standard error of the mean.
D. Now, you need to subtract the Mean Ct value of the POI from the Mean Ct
value of the HKG (of the same cDNA concentration). You also need to select
the highest standard error value between the POI and the HKG.
Example of Graph Pad Column Statistics Results:
X Labels
X Labels
X
Number
of values
A
B
HKG HKG
1ng
2ng
Y
Y
2
2
C
HKG
3ng
Y
2
D
HKG
4ng
Y
2
E
HKG
5ng
Y
2
F
POI
1ng
Y
2
G
POI
2ng
Y
2
H
POI
3ng
Y
2
I
POI
4ng
Y
2
J
POI
5ng
Y
2
Minimum 18.10 17.82 17.39 17.12 17.12 18.01 17.37 17.17 16.97 16.75
25%
Percentile
Median
18.11 17.83 17.47 17.26 17.12 18.07 17.49 17.18 17.01 16.82
75%
Percentile
Maximum 18.12 17.83 17.54 17.40 17.12 18.12 17.61 17.19 17.05 16.89
Mean
Std.
Deviation
Std. Error
18.11 17.83 17.47 17.26 17.12 18.07 17.49 17.18 17.01 16.82
0.014 0.007 0.106 0.198 0.0
0.077 0.169 0.014 0.056 0.098
Lower
95% Cl
Upper
95% Cl
17.98 17.76 16.51 15.48 17.12 17.37 15.97 17.05 16.50 15.93
0.010 0.005 0.075 0.14
0.0
0.055 0.12
0.01
0.04
0.07
18.24 17.89 18.42 19.04 17.12 18.76 19.01 17.31 17.52 17.71
0
0.301
0.477
0.602
0.699
E. From this table, you need to find the difference between the HKG and POI
(always use a positive value), plus select the highest standard error of the
mean values.
1ng: 18.11 – 18.07 = 0.04
SEM = 0.055
2ng: 17.83 – 17.49 = 0.34
SEM = 0.12
3ng: 17.47 – 17.18 = 0.29
SEM = 0.075
4ng: 17.26 – 17.01 = 0.25
SEM = 0.14
5ng: 17.12 – 16.82 = 0.30
SEM = 0.07
F. Now enter these values, along with the log of the concentrations, into another
Graph Pad Prism table to determine the slope. A slope of < 0.1 indicates a
good primer/cDNA concentration pairing. In Graph Pad Prism select “File,
New Project, Choose Type of Graph” and select the second graph option from
left. Below the picture of the graph, you can leave the “replicates” at default
and choose from the drop down menu on the right “Mean, Standard Error.”
G. In the first column of this chart (which is the X column) enter the log values
of the cDNA concentration. In the second and third column, enter the
corresponding Mean and Standard Error of the mean.
X Values
A
X Title
Title
X Mean
Mean
SEM
0.04
0.055
0.34
0.12
0.29
0.075
0.25
0.14
0.30
0.07
H. Select “Insert, New Analysis, Curves and Regression, Linear Regression,
Create.” Select “OK” and view your slope. In this instance, the slope is
0.3072 which is too high. You can delete up to two rows of values in an
attempt to get a better slope. Just make sure to keep 3 values. If I erase the
top two values (for 1ng and 2ng) in this example, my slope goes to 0.02785
which is good.
4. If your slope value is good (<0.1), then you can proceed with real time using any
concentration of cDNA that was used to determine the slope. In the case above, I
could use 3ng, 4ng, or 5ng.
5. If your slope value is not good (>0.1), then you can try the experiment again with
more or less cDNA. If you are still unable to get good results, double-check your
NTC and RT- values to see if maybe you have DNA contamination. There’s also
the possibility of primer dimers which would make your RT- Ct values lower. If
the primers won’t work, design new ones and begin again.