Wednesday, July 29, 2015

7 29 2015 EF1d qPCR 1 & 2

In an effort to find a better normalizing primer than the Actin one I have, I developed 4 possible normalizing primers. Today I ran two replicates of EF1d with a 1:9 dilution of cDNA. 


1682EF1d_FWDGAACTGCCCACTGATTTGCCJH7/22/2015205560O.luridaElongation factor 1-delta (EF-1-delta)A5D989
1681EF1d_REVTGTGGGGTGAAACACGTTGAJH7/22/2015205060O.luridaElongation factor 1-delta (EF-1-delta)A5D989
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

 Melt Curve

 Melt Curve


The amplification curves in both runs look great. There's no amplification in either set of NTCs. Also the latest amplifying samples were they same in both replicates. I then compared the replicates with the Opticon Correction to see how close are they. 

There is less than a 2 ct difference between the replicates with correction. The there is one major outlier which is likely caused by mechanical error. 

I will tomorrow I will average the Ct values and efficiencies to develop data to normalize expression data for the other targets. Also I've been reading on how to deal with outliers. If I throw out the data, then it screws up the statistics. One way I've seen this dealt with in the literature is to assume that outliers are caused by mechanical/human error and replace the values with the latest Ct value and efficiency found in the data which assumes the lowest observed expression. 

You can see paper here:

Differential gene expression profiling of Listeria monocytogenes in Cacciatore and Felino salami to reveal potential stress resistance biomarkers

You can see the raw data from the opticon and from the cfx

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