Showing posts with label Graphs. Show all posts
Showing posts with label Graphs. Show all posts

Wednesday, July 22, 2015

7 22 2015 HSPb11 qPCR 2

Today I ran duplicates for the targets I ran last week. I'm hoping to get strong replicates for analysis. Here I ran the HSPb11 which last week had issues with the NTC. 

Primers:
1650Hspb11_FWDATGTTTCCTGGTCTCCGTCAJH5/21/20152055O.luridaHeat shock protein beta-11 (Hspb11) (Placental protein 25) (PP25)Q9Y547
1649Hspb11_REVCATCAACGCCAGGGGAACTTJH5/21/20152055O.luridaHeat shock protein beta-11 (Hspb11) (Placental protein 25) (PP25)Q9Y547
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
Amp
Melt

NTCs
Amp
Melt

The amplification and melt curves look good. There's no amplification in the NTCs. To analyze this data I ran it through a script to do stats and make some graphs. 

Expression Bar Graph

Statistics

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

Terms:
                         Pop        Treat    Pop:Treat    Residuals
Sum of Squares  1.913763e-14 3.460040e-15 6.197430e-15 2.482985e-13
Deg. of Freedom            2            1            2           42

Residual standard error: 7.688868e-08
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
              p adj
N-H  0.6743300
S-H  0.1825199
S-N  0.6131938

$Treat
               p adj
T-C  0.4485315

$`Pop:Treat`
                   p adj
N:C-H:C   0.9968662
S:C-H:C   0.4617887
H:T-H:C  0.9999847
N:T-H:C   0.9924229
S:T-H:C   0.9930620
S:C-N:C   0.7546118
H:T-N:C  0.9879773
N:T-N:C   0.9999986
S:T-N:C   0.9999992
H:T-S:C  0.3687373
N:T-S:C  0.8077927
S:T-S:C  0.8022228
N:T-H:T   0.9771056
S:T-H:T   0.9785530
S:T-N:T  1.0000000

The Stats show there are no significant differences between the treatment or controls or populations which can be seen in the expression graphs and boxplot. 

This like the BMP2 data shows there are huge differences in the stats between the reps. I re ran the previous data without removing the outliers to see how it changes the data. 

Expression Bargraph

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

Terms:
                         Pop        Treat    Pop:Treat    Residuals
Sum of Squares  3.811151e-19 2.405560e-20 3.873500e-21 1.510927e-18
Deg. of Freedom            2            1            2           42

Residual standard error: 1.896693e-10
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
                p adj
N-H   0.0145606
S-H   0.9658692
S-N  0.0273635

$Treat
               p adj
T-C  0.418125

$`Pop:Treat`
                    p adj
N:C-H:C   0.4354280
S:C-H:C   0.9999831
H:T-H:C  0.9888451
N:T-H:C   0.5629155
S:T-H:C  0.9980358
S:C-N:C  0.5358964
H:T-N:C  0.1506397
N:T-N:C  0.9999464
S:T-N:C  0.2184309
H:T-S:C  0.9688693
N:T-S:C   0.6656163
S:T-S:C  0.9911381
N:T-H:T   0.2232571
S:T-H:T   0.9999602
S:T-N:T  0.3113530


Even with the outliers the expression changes enough that stats still show there being a significant difference between the oyster bay population and the other two sites. This can be seen with the boxplot below. 


For comparison sake I pulled out the Ct values from qpcR for both the CFX and Opticon data to compare to the Ct values generated by the Opticon and CFX programs. Below are some line graphs produced from the Ct data. 

All

CFX/CFXqpcR
Opticon/OptiqpcR
Opticon/CFX
OptiqpcR/CFXqpcR

You can see that the Opticon and OptiqpcR values don't match. The qpcR values are somewhat deflated compared to the Opticon ct values. The CFX and CFXqpcR data looks much closer with only some small differences between the read outs. 

It looks like the qpcR package doesn't handle the raw fluorescence data from the Opticon as well as it does with the CFX data. This suggests that the CFX may be the better machine. 

Sadly this doesn't fix the issue between the reps as the BMP2 data was run on the same machine. I'm unsure what to do at this point. 

You can see the raw data for the qPCR runs last week here and this week here. You can also see the Ct value comparison CSV here. 
Posted by at No comments:
Labels: , , ,

7 22 2015 BMP2 qPCR 2

Today I ran duplicates for the targets I ran last week. I'm hoping to get strong replicates for analysis. Here I ran the BMP2 which last week had issues with the NTC. 

Primers:
1640BMP-2_FWDTGAAGGAACGACCAAAGCCAJH5/21/20152055O.luridaBone morphogenetic protein 2 (BMP-2) (Bone morphogenetic protein 2A) (BMP-2A)P12643
1639BMP-2_REVTCCGGTTGAAGAACCTCGTGJH5/21/20152055O.luridaBone morphogenetic protein 2 (BMP-2) (Bone morphogenetic protein 2A) (BMP-2A)P12643
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

These amplification curves look better than the last run of BMP2. There is some minor amplification in the NTCs but it is larger than the target and is probably an artifact of the PCR process. To analyze these data I ran them through an updated stats and graphs script which runs a Two Way ANOVA and produces a boxplot and expression bar graph. The data did not have its outliers eliminated which worked fine with this as most samples showed some expression. 

Expression Bar Graph


Statistics

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

Terms:
                         Pop        Treat    Pop:Treat    Residuals
Sum of Squares  1.703293e-20 3.032145e-20 2.201442e-20 6.077281e-20
Deg. of Freedom            2            1            2           42

Residual standard error: 3.803908e-11
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  0.4089519
S-H  0.0041846
S-N  0.1002050

$Treat
             diff           lwr           upr    p adj
T-C  4.14e-05

$`Pop:Treat`
                 diff           lwr           upr     p adj
N:C-H:C   0.6348257
S:C-H:C   0.0001275
H:T-H:C  0.9973637
N:T-H:C  0.9999701
S:T-H:C  0.9814628
S:C-N:C   0.0134535
H:T-N:C  0.3564950
N:T-N:C  0.5192134
S:T-N:C  0.2393859
H:T-S:C  0.0000292
N:T-S:C  0.0000710
S:T-S:C  0.0000133
N:T-H:T   0.9997768
S:T-H:T  0.9998827
S:T-N:T  0.9953619

There definitely appears a significant difference between treatment and control. From the stats it appears the Oyster Bay population control has significantly larger expression of BMP2 than the other two populations as well as it the Oyster Bay treatment. You can see this in the bar graph generated below.


The most distressing thing about these data is that it doesn't match the graphs from last week. The controls have much lower expression than they did last week which should not have changed between replicates. I reran the previous data with all the outliers but it still doesn't change the statistics. 

Previous Expression Data

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

Terms:
                         Pop        Treat    Pop:Treat    Residuals
Sum of Squares  3.918260e-22 9.723005e-21 1.573763e-21 9.279358e-21
Deg. of Freedom            2            1            2           38

Residual standard error: 1.56267e-11
Estimated effects may be unbalanced
4 observations 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   0.4224591
S-H   0.8174685
S-N  0.8189700

$Treat
         p adj
T-C  2e-07

$`Pop:Treat`
                    p adj
N:C-H:C   0.6961640
S:C-H:C  0.9298189
H:T-H:C  0.0032438
N:T-H:C  0.0048368
S:T-H:C  0.2481527
S:C-N:C  0.1806352
H:T-N:C  0.0000405
N:T-N:C  0.0000732
S:T-N:C  0.0140727
H:T-S:C  0.0411016
N:T-S:C  0.0532668
S:T-S:C  0.7297433
N:T-H:T   1.0000000
S:T-H:T   0.7788719
S:T-N:T   0.7991437

The stats don't match which is distressing. Even the boxplots dont seem to match. 

You can see the raw data files for this week here and last week here. 

Posted by at No comments:
Labels: , , ,
Subscribe to: Posts (Atom)