7 15 2015 H2A qPCR

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 third is the H2A target. You can read about the first (H3.3) here and the second here. I did not run replicates because I've found that with the current procedure the replicates typically are very close.

Primer:

1606	H2A_FWD	GCTGGGGTTTTTCTGGGTCT	JH	5/21/2015	20	55		O.lurida	Histone H2A	P02270
1605	H2A_REV	GGAACTACGCCGAGAGAGTG	JH	5/21/2015	20	55		O.lurida	Histone H2A	P02270

Reagent Table:

	Volume	Reactions X116
Ssofast Evagreen MM	10	1160
FWD Primer	0.5	58
REV Primer	0.5	58
1:9 cDNA	9

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

Program:

Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	60 C	1 min
Read
	72 C	30 sec
Read
Repeat Elongation 39 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	55 - 95 C	30 sec
	21 C	10 min
End

Plate Layout:

1	2	3	4	5	6	7
DNased 42215 HC1	DNased 42215 NC1	DNased 42215 SC1	DNased 42215 HT1 1	DNased 42215 NT1 1	DNased 42215 ST1 1	NTC
DNased 42215 HC2	DNased 42215 NC2	DNased 42215 SC2	DNased 42215 HT1 2	DNased 42215 NT1 2	DNased 42215 ST1 2	NTC
DNased 42215 HC3	DNased 42215 NC3	DNased 42215 SC3	DNased 42215 HT1 3	DNased 42215 NT1 3	DNased 42215 ST1 3	NTC
DNased 42215 HC4	DNased 42215 NC4	DNased 42215 SC4	DNased 42215 HT1 4	DNased 42215 NT1 4	DNased 42215 ST1 4	NTC
DNased 42215 HC5	DNased 42215 NC5	DNased 42215 SC5	DNased 42215 HT1 5	DNased 42215 NT1 5	DNased 42215 ST1 5
DNased 42215 HC6	DNased 42215 NC6	DNased 42215 SC6	DNased 42215 HT1 6	DNased 42215 NT1 6	DNased 42215 ST1 6
DNased 42215 HC7	DNased 42215 NC7	DNased 42215 SC7	DNased 42215 HT1 7	DNased 42215 NT1 7	DNased 42215 ST1 7
DNased 42215 HC8	DNased 42215 NC8	DNased 42215 SC8	DNased 42215 HT1 8	DNased 42215 NT1 8	DNased 42215 ST1 8

Results:

All samples

NTCs

These runs look good as well but with a dimer appearing in 1 of the 4 NTCs. This is ok because it is much smaller than the target gene and thus not a sign of contamination. To determine if this was useful for normalizing I ran an ANOVA and Tukey's HSD to determine significant differences between treatment and controls. 

Call:

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

Terms:

                         Pop        Treat    Pop:Treat    Residuals

Sum of Squares  5.114600e-24 4.006829e-22 1.928451e-22 2.578036e-21

Deg. of Freedom            2            1            2           35

Residual standard error: 8.582435e-12

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)

$Pop

             diff           lwr          upr     p adj

N-H  0.9770758

S-H   0.9980605

S-N   0.9652703

$Treat

            diff          lwr          upr     p adj

T-C  0.0258271

$`Pop:Treat`

                 diff           lwr          upr     p adj

N:C-H:C   0.9284828

S:C-H:C   0.9913900

H:T-H:C   0.1254093

N:T-H:C   0.8827253

S:T-H:C   0.4351436

S:C-N:C  0.9989003

H:T-N:C   0.7722933

N:T-N:C   0.9999997

S:T-N:C   0.9823553

H:T-S:C   0.4682695

N:T-S:C   0.9968805

S:T-S:C   0.8575230

N:T-H:T  0.7753255

S:T-H:T  0.9778512

S:T-N:T   0.9865781

This would not make a good normalizing gene as there is a significant difference between the treatment and control. There doesn't appear to be any significant differences between populations or between combinations of treatments and controls. This also isn't very interesting because of the lack of clear differences between populations. 

You can see the raw data here.