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

Wednesday, June 24, 2015

6 24 2015 qPCR Quality Check

Today I ran a qPCR quality check to determine if our Opticon two is producing valid data. I did this because the other day we noticed a discrepancy with the data that showed samples in the top and bottom rows had lower expression than those in the middle rows. To test if the machine was working properly I made a master mix including a template of isolated gDNA that I know amplifies and equally distributed it across the plate.

Primer:
1505Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
1504Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 

Reagent Table:
VolumeReactions X52
Ssofast Evagreen MM 10520
FWD Primer0.526
REV Primer0.526
Nuclease Free H2O8416
gDNA152

The sample I used was from Oly seed oyster isolations I did a while back. 
Sample Info:
Sample IDDateTimeng/ul
NF205/7/201511:17 AM876.31

To make the master mix I added all the reagents together from greatest volume to least volume and then added the gDNA template. I vortexed briefly to ensure homogeneous mixture. I then pipetted 20 ul of this mixture into every other well to make a checker board pattern on the plate. 

Plate Layout:
123456789101112
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample
SampleSampleSampleSampleSampleSample

This pattern was to test the machines fluorescence reading capabilities across the entire plate without having to fill every well with sample. 

I ran the following program which is the same for all the other qPCR's that I have been running.

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

Results:
Amplification
Melt Curve
 Plate Fluorescence

As you can see from the amplification, while all of the well should have had exactly the same amplification there was a significant difference between wells with some wells being orders of magnitude higher. The melt curve shows similar. My favorite image though is the plate fluorescence which shows a rough estimate of the fluorescence detected in each well by the machine. Clearly the center wells have higher fluorescence detection than wells along the edges of the plate. At this time it is apparent that we need to recalibrate the Opticon as it clearly is not producing valid data.

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6 24 2015 PCR Miner with Friedman lab data

After running the qPCRs in the Friedman lab yesterday, I have run them through PCR miner. I then took the results and normalized the expression values of TLR2.1. I'll go through the steps here.

First I need to export the data from BioRad CFX manager.

  1. Click Export
  2. Go to Export All Data Sheets
  3. Select CSV as export file type
This causes a ton of CSV's to be produced most of them have absolutely no value for PCR Miner. The one we need is titled: Quantification Amplification Results _ FAM. FAM denotes the luminescence frequency which picked up the lumination from the SYBR green dye. 

Now we need to format this file.
  1. Rename each column with the appropriate Gene, Replicate, and sample Number.
Now you just need to copy the data into the entry area of PCR Miner.


PCR Miner pops out the data which you will download from the following screen:



Then this text file can be opened in Excel. Once in excel I highlighted the Cycle threshold value and the efficiency for each population. Then I created the expression value with the equation =1/(1+Efficiency)^CycleThreshold. I did this with both the TLR2.1 and Actin genes. Finally I divided the TLR2.1 expression values by the Actin expression values to get the normalized expression value of TLR2.1. 


This is just the first full run using 8 samples per population per treatment but we can see some interesting trends. Expression of TLR2.1 is nearly doubled in the Fidalgo population (N) and tripled in the Oyster Bay population (S) due to Heat Stress. The values for the Dabob population are a little dubious due to the lack of expression from about half the population. Though it does show that while expression seems to be down regulated, it is roughly the same between control and treatment.

You can see the raw fluoresence file here, the PCR Miner output here, and my analyzed excel file here.
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6 23 2015 Actin qPCR 2

Yesterday I re ran the Actin qPCR to create a normalizing reference for the Friedman Lab's qPCR Machine.

Primers:
1505Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
1504Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 

Reagent Table:
VolumeReactions X58
Ssofast Evagreen MM 10580
FWD Primer0.529
REV Primer0.529
Nuclease Free H2O8464
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
  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:


NTCs


The actin primer showed relatively equal expression across samples which is what we expected since we're using a properly calibrated machine. There were no miss expression values for samples which means it was expressed in all populations. I ran this and the TLR2.1 Data through PCR Miner to analyze expression. I will talk about that in my next post. 

You can find the raw data file here.
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6 23 2015 TLR2.1 qPCR

Yesterday after discovering that the Opticon was acting up, I decided to run a couple qPCR's on the Biorad CFX in the Friedman lab. The first was the TLR2.1 gene which had not been present in the Dabob population during the initial primer checks.

Primer:
1630TLR2.1_FWDACAAAGATTCCACCCGGCAAJH5/21/20152055O.luridaToll-like receptor 2 type-1Q9DD78
1629TLR2.1_REVACACCAACGACAGGAAGTGGJH5/21/20152055O.luridaToll-like receptor 2 type-1Q9DD78

Reagent Table:
VolumeReactions X58
Ssofast Evagreen MM 10580
FWD Primer0.529
REV Primer0.529
Nuclease Free H2O8464
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
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



So the graph doesn't really do justice for the results. There is amplification in a majority of samples but interestingly only about half of the Dabob population shows expression of this allele. The other half show absolutely no expression. There are a few individuals which show a very low expression of this allele but others show some of the highest expression. Overall this is pretty confusing and may be indicative that the Dabob population is more heterogeneous for this allele than the other populations. After running this qPCR, I ran another Actin qPCR using the friedman machine to create a normalizing standard which I will talk about in my next post.

You can view the raw data here.
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Tuesday, June 23, 2015

6 22 2015 Actin qPCR

Last night I ran a qPCR on Actin using the 48 cDNA samples produced by Sam. I ran it using the same procedure as I did for the CARM samples yesterday morning so that I can use it as a normalizing gene.

Primer Used:
1505Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
1504Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 

Master Mix Reagent Table:
VolumeReactions X58
Ssofast Evagreen MM 10580
FWD Primer0.529
REV Primer0.529
Nuclease Free H2O8464
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
qPCR Program:
Sybr New Plate+Sybr cDNA 60 melt 2 Read
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:

Amplification

 Melt Curve

As you can see the amplification and melt curves look good. Upon closer examination I did notice however that the rows at the top and bottom had much lower amplification than those in the middle. This is concerning as it could be giving me false values for samples in these spots. I've decided to forego using the Opticon and have now run samples on the BioRad in the Friedman lab in FSH. 

You can see the raw data file here.
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