Thursday, July 16, 2015

7 16 2015 HSP70 qPCR

Today I started running more targets that may be of interest due expected changes in expression. Here I explore HSP70 which is expected to have increased expression due to exposure to increased temperatures. 

Primers:
HSP70 
FWD - TTGTCGCCATTTTCCTCGCT
REV - GTTCCGATTTGTTCCGTGCC

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
Program:
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
Read
72 C30 sec
Read
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
End
Plate Layout:
1234567
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
Results:

All samples

NTCs

This looks interesting in terms of expression, there is some amplification in the NTCs but it was a product much larger than the target. To see if there are any interesting expression levels I ran this through an ANOVA and Tukey's HSD to produce any significant differences between the treatment, control, and populations. 


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

Terms:
                         Pop        Treat    Pop:Treat    Residuals
Sum of Squares  7.777300e-20 1.174464e-19 7.510100e-20 2.988660e-18
Deg. of Freedom            2            1            2           41

Residual standard error: 2.699892e-10
Estimated effects may be unbalanced
1 observation deleted due to missingness
> TukeyHSD(fit)
  Tukey multiple comparisons of means
    95% family-wise confidence level

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

$Pop
            p adj
N-H  0.6768333
S-H  0.6234033
S-N  0.9972407

$Treat
              p adj
T-C  0.2116992

$`Pop:Treat`
                   p adj
N:C-H:C   0.9999993
S:C-H:C  1.0000000
H:T-H:C  0.9999997
N:T-H:C   0.8823690
S:T-H:C   0.8051192
S:C-N:C  0.9999986
H:T-N:C  0.9999855
N:T-N:C   0.9123072
S:T-N:C   0.8463452
H:T-S:C  0.9999999
N:T-S:C   0.8771414
S:T-S:C   0.7981469
N:T-H:T   0.8524787
S:T-H:T   0.7660122
S:T-N:T   0.9999953

There's no significant differences between anything which is very surprising as I would expect at least a strong difference between treatment and control. Steven has suggested that this primer may not actually be for HSP70 since we did not develop it. These results seem to confirm it though. The expression graph produced from the expression data doesn't seem to be of any help either. I think I may need to go redevelop some primers for HSP70 to hopefully get one that works. 


You can see the raw data here.

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