I worked on figuring out some of the stats for my qPCR data with Shelly on Thursday. I decided to go with an ANOVA/Tukey’s Honest Significance Difference Test, with the data being normalized using a log transform. Final qPCR plots are attached below–capital letters indicate differences in treatment, asterisks denote differences between individual groups, and capital letters next to ploidy key indicate differences between diploids and triploids.
To account for N/A Cq values, I substituted in a Cq value of 45 wherever the Cq value was nonexistent. This allows for some initial analysis of the data to see which genes might warrant further work. Below are the adjusted qPCR plots. I also generated the COX1 Cq plot (not normalized to actin) to examine relative mitochondrial abundance between stressed and non-stressed diploids and triploids.
I ran SOD and ATP Synthetase qPCRs last week. Currently, I’m working on getting box plots worked out with the R script–the main issue I’m trying to sort out is how to account for the N/A values for some of the samples as the current plots just ignore it. I’m also adding a statistical analysis portion to the script so that we can start visualizing which treatments are statistically different.
Link to ATP Synthetase qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2019-01-23%2014-56-33_BR006896.pcrd
Link to SOD qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2019-01-09%2015-08-24_BR006896.pcrd
Today, I made a GitHub account and generated box plots for all my qPCR data using R. Attached below are pictures of the box plots:
I ran a qPCR assay on my desiccated + elevated temp. samples with HSC70, MBD2, and MeCP2 as my gene targets. For protocol used, please refer to previous notebook entries.
Link to Excel spreadsheet of Cq values: https://docs.google.com/spreadsheets/d/1AhdJLEcXt6vnNBcGVLnc926KlZ5J-Nt8JMpiCdrUvaU/edit?usp=sharing
Link to HSC70 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-19%2016-24-55_BR006896.pcrd
Link to MBD2 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-20%2013-17-34_BR006896.pcrd
Link to MeCP2 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-20%2014-58-17_BR006896.pcrd
In the last two qPCR assays, no fluorescence was detected. Turns out we were using Promega master mix which is specifically for probe-based assays…as such, it doesn’t have any fluorescent dye in it, explaining the lack of fluorescence detection in any of the assays. I switched over to using a different master mix that should solve the issue.
I reran the DNMT1 primer assays and will run an actin qPCR to determine if it might be a possible normalizing gene. For protocol used, please refer to previous lab book entries.
Link to Excel spreadsheet of Cq values for both actin and DNMT1: https://docs.google.com/spreadsheets/d/1AhdJLEcXt6vnNBcGVLnc926KlZ5J-Nt8JMpiCdrUvaU/edit?usp=sharing
Link to DNMT1 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-12%2015-24-28_BR006896.pcrd
Link to actin qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-14%2016-16-16_BR006896.pcrd
All replicates are in adjacent wells. The order of the samples in the well plate by rows is: D01, D02, D03, D04, D05, D06, D07, D08, D09, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, T11, T12, T13, T14, T15, T16, T17, T18, T19, T20
Before the long weekend, I ran a qPCR assay with my desiccated + elevated temp. samples. There were 40 samples in total and I ran one plate with DNMT1 (DNA methyltransferase) as my gene target.
The plate is laid out with duplicates in adjacent wells and the order of samples is as follows:
D01, D02, D03, D04, D05, D06, D07, D08, D09, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, T11, T12, T13, T14, T15, T16, T17, T18, T19, T20
There are 2 positive controls with gDNA in wells G9 and G10. There is a no-template control (negative control) with no template in wells G11 and G12.
To run the qPCR assay, I created a mastermix with 20 µL of forward primer, 20 µL of reverse primer, 400 µL of 2x qPCR master mix, and 320 µL of DEPC-treated water. 19 µL of the mastermix was put into each well and a subsequent 1 µL of cDNA was put in for each sample (water for the negative controls and gDNA for the positive controls). Note that all sample cDNA used was a 1:5 dilution.