2/12-2/16: Primer Validations

Introduction: 

 

Primer validation is important to ensure that the primers we will be working with in our qPCR experiment with D. aquaticus are functioning correctly. Primer validation must be done before performing our test to see what is considered cold shock or heat shock. The plan is to validate 6 different primers and perform qPCR on them to see if the results are accurate. For each primer, 5 different dilutions are done, 1:1, 1:5, 1:25, 1:125, and 1:625. With these 5 dilutions, we are able to compare the 5 graph lines on qPCR to see if they line up, and if this is the case, there will be a efficiency value between 90-110%.

 


Methods:

 

Nanodrop values were taken on our RNA extraction products, the values are shown in figure 2. 

 

The protocol for our RT qPCR experiment is shown below. 

 


Figure 1: Luna 1 step RT qPCR kit protocol. 

 

Results from the qPCR were finalized 2/12 and the data was validated in excel to determine if the primer validation was successful.

 

 

Results: 

 

Tube

ng/ul

A260/A280

A260/A230

1

1795.9

2.08

2.25

2

2385.4

2.12

2.32

Figure 2: Nanodrop results on our RNA samples, taken 2/12.

 

 

Tube

Cq 1

Cq 2

Cq 3

Avg Cq

Sample Quantity 

Log (sample quantity)

A

17.39

16.95

16.83

17.05666667

1

0

B

19.5

19.27

19.27

19.34666667

0.2

-0.698970004

C

21.59

21.59

21.73

21.63666667

0.04

-1.397940009

D

24.44

24.26

24.66

24.45333333

0.008

-2.096910013

E

26.97

26.45

26.7

26.70666667

0.0016

-2.795880017

Figure 3: Cq values and log values of each run of qPCR. 

 

 


Figure 4: Graph demonstrating the relationship between average Cq values and log values. The slope equation is shown below the trendline.



Discussion:

 

After plugging in all of the Cq values into excel and completing the required equations, the efficiency was 93.36811752, or ~93%. This would support that the primer validation was successful, because we want an efficiency percentage to be between 90-110%. The primers used seem to be in good condition to be used for the future testing with cold and heat shock.

 

 





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