qPCR for Gene Expression


This week we were finally able to do qPCR. We didn't have lab Monday, so we started Tuesday with diluting our samples to prep for RNA isolation. We tried to get as close as possible to an OD600 of 1. After a bit of work, we ended up with the closest dilutions prier to RNA isolation that we've had so far. 

Before Dilutions:
T1  2.86
T2  2.14
T3  2.73
C1  3.06
C2  3.20
C3  3.11 

After Dilutions:
T1  0.99
T2  1.01
T3  0.99
C1  0.98
C2  1.01
C3  0.99


Wednesday we did RNA isolation and cDNA synthesis. After RNA our samples we nanodropped the samples to see our concentration. 

       ng/μl      A260/280   A260/230
T1  6.6         1.83            1.08
T2  16.3       1.97            1.08
T3  32.6       2.06            1.35
C1  35.3       1.96            1.09
C2  24.2       2.00            1.20
C3  23.9       1.89            0.81

After that we did cDNA synthesis. We did our best to load 92ng of RNA for each sample.

T1 14uL - 92.4ng
T2 5.7uL - 92.9ng
T3 2.8uL - 91.28ng
C1 2.6uL - 91.78ng
C2 3.8uL - 91.96ng
C3 3.9uL - 93.21ng

After that we proceeded with cDNA synthesis.


Thursday we loaded the qPCR plate in preparation for the qPCR. We started by diluting our cDNA stock 
1 in 10, for 50 μl (so 5 μl stock and 45 μl nuc free water). 

We loaded the plates, starting with 6 μl of the dye and mastermix. After fully pipetting on the needed wells, we than did the 4 μl of 1/10 diluted sample. When loading the wells, we propped the plate up on a tube holder in order to get the plate at an angle for ease during pipetting. We loaded the wells going down the columns for the mastermix, and across the rows for the samples.

 

 pfs

 SecA

 Gap3

 

 T1

 T1+pfs

T1 +pfs

 T1+pfs

 T1+SecA

 T1+SecA

 T1+SecA

 T1+Gap3

 T1+Gap3

 T1+Gap3

 

 

 

 T2

 T2+pfs

 T2+pfs

 T2+pfs

 T2+SecA

 T2+SecA

 T2+SecA

 T2+Gap3

 T2 +Gap3

 T2+Gap3

 

 

 

 T3

 T3+pfs

 T3+pfs

 T3+pfs

 T3+SecA

 T3+SecA

 T3+SecA

 T3+Gap3

 T3+Gap3

 T3+Gap3

 

 

 

 C1

 C1+pfs

 C1+pfs

 C1+pfs

 C1+SecA

 C1+SecA

 C1+SecA

 C1+Gap3

 C1+Gap3

 C1+Gap3

 

 

 

 C2

 C2+pfs

 C2+pfs

 C2+pfs

 C2+SecA

 C2+SecA

 C2+SecA

 C2+Gap3

 C2+Gap3

 C2+Gap3

 

 

 

 C3

 C3+pfs

 C3+pfs

 C3+pfs

 C3+SecA

 C3+SecA

 C3+SecA

 C3+Gap3

 C3+Gap3

 C3+Gap3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 Control

  pfs

  pfs

  pfs

 SecA

 SecA

 SecA

 Gap3

 Gap3

 Gap3

 

 

 


After loading the plate, we moved the plate into the centrifuge. Unfortunately the plate cover wasn't applied correctly, and when loaded into the centrifuge the samples went everywhere. 

Luckily enough we still had enough sample, so we tried again Friday, repeating the process of loading the plates. This time the plate cover was applied much better, and we moved on to the actual qPCR. 


red is SecA, green is Gap3 and blue is pfs

 






After looking at the data, we saw that the difference between the pfs tests and controls for gene expression weren't much difference. Looking at the actual math, any difference between the numbers has to be attributed natural varience, not do the the hydrogen peroxide. Instead moving forward we will target LuxS and KatA. 

LuxS will be our new AMC pathway gene, while the KatA will show if the hydrogen peroxide is affecting our cells. If needed, we will up our hydrogen peroxide concentration or change our stressor. 


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