Wednesday, July 15, 2015

7 15 2015 H3.3 qPCR run normalizing gene test

Since I don't have a good normalizing gene I decided to check to see if I can create one with the primers that I have. When I ran the initial checks on histone targets, they were expressed in very similar levels across all treatments. The first is the H3.3 target. I did not run replicates because I've found that with the current procedure the replicates typically are very close.

1610H3.3_FWDCACGCTCTCCTCGAATCCTCJH5/21/20152055O.luridaHistone H3.3Q6P823
1609H3.3_REVAAGTTGCCTTTCCAGCGTCTJH5/21/20152055O.luridaHistone H3.3Q6P823

Reagent Table:
VolumeReactions X116
Ssofast Evagreen MM101160
FWD Primer0.558
REV Primer0.558
1:9 cDNA9
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 11 ul Master Mix to each tube
  5. Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
72 C30 sec
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 min
Plate Layout:
DNased 42215 HC1DNased 42215 NC1DNased 42215 SC1DNased 42215 HT1 1DNased 42215 NT1 1DNased 42215 ST1 1NTC
DNased 42215 HC2DNased 42215 NC2DNased 42215 SC2DNased 42215 HT1 2DNased 42215 NT1 2DNased 42215 ST1 2NTC
DNased 42215 HC3DNased 42215 NC3DNased 42215 SC3DNased 42215 HT1 3DNased 42215 NT1 3DNased 42215 ST1 3NTC
DNased 42215 HC4DNased 42215 NC4DNased 42215 SC4DNased 42215 HT1 4DNased 42215 NT1 4DNased 42215 ST1 4NTC
DNased 42215 HC5DNased 42215 NC5DNased 42215 SC5DNased 42215 HT1 5DNased 42215 NT1 5DNased 42215 ST1 5
DNased 42215 HC6DNased 42215 NC6DNased 42215 SC6DNased 42215 HT1 6DNased 42215 NT1 6DNased 42215 ST1 6
DNased 42215 HC7DNased 42215 NC7DNased 42215 SC7DNased 42215 HT1 7DNased 42215 NT1 7DNased 42215 ST1 7
DNased 42215 HC8DNased 42215 NC8DNased 42215 SC8DNased 42215 HT1 8DNased 42215 NT1 8DNased 42215 ST1 8

All samples


The graphs really look great with little amplification in the NTCs that generate a produce larger than the target. 

To check to see if the target works as a normalizing gene I ran the raw fluorescence through the R script to see if there is any stastical differences in expression. 

   aov(formula = expression ~ Pop + Treat + Pop:Treat, data = rep2res2)

                         Pop        Treat    Pop:Treat    Residuals
Sum of Squares  2.815300e-18 5.718240e-18 2.010162e-17 5.558573e-17
Deg. of Freedom            2            1            2           42

Residual standard error: 1.150422e-09
Estimated effects may be unbalanced
> TukeyHSD(fit)
  Tukey multiple comparisons of means
    95% family-wise confidence level

Fit: aov(formula = expression ~ Pop + Treat + Pop:Treat, data = rep2res2)

            p adj
N-H   0.3728762
S-H  0.9752878
S-N  0.4934820

            p adj
T-C  0.0437989

                     p adj
N:C-H:C   0.0147134
S:C-H:C  0.9984848
H:T-H:C   0.9873039
N:T-H:C  0.9428648
S:T-H:C   0.9943919
S:C-N:C  0.0419768
H:T-N:C  0.0731341
N:T-N:C  0.0010668
S:T-N:C  0.0573493
H:T-S:C   0.9998977
N:T-S:C  0.7771138
S:T-S:C   0.9999946
N:T-H:T  0.6380232
S:T-H:T  0.9999981
S:T-N:T   0.7025093

Items in bold show significant differences. Sadly from these tests H3.3 is not good for gene normalization as the treatment and control are significantly different. Also the Fidalgo Control (NC) is significantly different from all other population controls and from Fidalgo Treatment (NT). 

On the positive side, this is great as it shows a significant different in populations at control points which changes due to heat shock. Now if I can only find a normalizing gene. 

You can see the raw data here.

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