--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left layout: post title: qPCR - C.gigas Ctenidia cDNA for Noah's Heat-Mechanical Stress Project date: '2023-07-19 15:40' tags: - qPCR - CFX Connect - CFX96 - 18s - GAPDH - VIPERIN - HSP70 - HSP90 - ATP synthase - citrate synthase categories: - 2023 - Miscellaneous --- Using [cDNA from 20230713](https://robertslab.github.io/sams-notebook/posts/2023/2023-07-13-Reverse-Transcription---C.gigas-RNA-from-Noahs-Heat-Mechanical-Stress-Project/), performed qPCR on the following primer sets selected by Steven: | SRID | Primer-name | |------|----------------------| | 1408 | Cg_18s_F | | 1409 | Cg_18s_R | | 1385 | Cg_ATPsynthase_F | | 1386 | Cg_ATPsynthase_R | | 1383 | Cg_citratesynthase_F | | 1384 | Cg_citratesynthase_R | | 1172 | Cg_GAPDH_205_F | | 1173 | Cg_GAPDH_355_R | | 599 | Cg_HSP70_F | | 598 | Cg_HSP70_R | | 1532 | Cg_Hsp90_F | | 1533 | Cg_Hsp90_R | | 1829 | Cg_VIPERIN_F | | 1828 | Cg_VIPERIN_R | qPCR calcs: - [20230719 - qPCR Calcs Noah gigas ctenidia cDNA](https://docs.google.com/spreadsheets/d/1DXVSlQtKJsmwNEUbx-IOEdoOFy8FwFqmw8n7LW3pqgw/edit#gid=0) (Google Sheet) Reactions were run on white, low-profile, 96-well qPCR plates (USA Scientific). A total of two plates were run. One was run on a CFC Connect (BioRad) and the other on a CFX96 (BioRad). See the qPCR Report in the [RESULTS](#results) section below for cycling params, plate layouts, etc. All samples were run in _singular_. Normally duplicates/triplicates are run. However, due to time and sample constraints for Noah's project/poster, samples were just run in _singular_. No template controls were run in duplicate. --- # RESULTS Here's a brief summary of the qPCR data, based on looking at the [amplification and melt curve plots below](#plots). `18s`: No Template Controls (NTCs) show contamination in melt curve and will need to be rerun. `ATP_synthase`: Melt curve looks good. Amplification looks good. `Citrate_synthase`: Melt curve looks good. Amplification looks good. `GAPDH`: NTC's do show some very late amplification, but does not cross Cq threshold. Amplification plots show distinct separation between groups of samples, suggesting some response to treatments. Needs to be explored further. `HSP70`: Amplification looks good. Melt curves are noticeably more broad than other primer sets. Additional scrutiny reveals a potential additional peak. Both aspects suggest that more than one product is being amplified. This is not terribly surprising, as there are numerous HSP70 present in organisms and they share a great deal of sequence homology to one another. Without careful primer design, it's very easy to end up amplifying more than one gene in the HSP70 family. `HSP90`: Melt curve looks good. Amplification looks good. `VIPERIN`: Melt curve looks good. Amplification looks good. --- qPCR Results file (CSV) - [sam_2023-07-19_11-53-35_Connect-Quantification-Cq-Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19_11-53-35_Connect-Quantification-Cq-Results.csv) qPCR Data file (QPCRD - CFX Maestro required) - [sam_2023-07-19 2011-53-35_Connect.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2023-07-19%2011-53-35_Connect.pcrd) qPCR Report (PDF) - [sam_2023-07-19 2011-53-35_Connect.pdf](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2023-07-19%2011-53-35_Connect.pdf) qPCR Results file (CSV) - [sam_2023-07-19 2011-54-14_CFX96-Quantification-CqResults.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Quantification-CqResults.csv) qPCR Data file (QPCRD - CFX Maestro required) - [sam_2023-07-19 2011-54-14_CFX96.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2023-07-19%2011-54-14_CFX96.pcrd) qPCR Report (PDF) - [sam_2023-07-19 2011-54-14_CFX96.pdf](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2023-07-19%2011-54-14_CFX96.pdf) --- #### PLOTS NOTE: NTCs are denoted by red lines in all plots. | Target | Amplification | Melt Curve | |--------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------| | Cg_18s | ![Cg_18s amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_18s-amp-plots.png) | ![Cg_18s melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_18s-melt-plots.png) | | Cg_ATPsynthase | ![Cg_ATPsynthase amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_ATPsynthase-amp-plots.png) | ![Cg_ATPsynthase melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_ATPsynthase-melt-plots.png) | | Cg_citratesynthase | ![Cg_CitrateSynthase amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_CitrateSynthase-amp-plots.png) | ![Cg_CitrateSynthase melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_CitrateSynthase-melt-plots.png) | | Cg_GAPDH | ![Cg_GAPDH amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_GAPDH-amp-plots.png) | ![Cg_GAPDH melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-53-35_Connect-Cg_GAPDH-melt-plots.png) | | Cg_HSP70 | ![Cg_HSP70 amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Cg_HSP70-amp-plots.png) | ![Cg_HSP70 melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Cg_HSP70-melt-plots.png) | | Cg_Hsp90 | ![Cg_HSP90 amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Cg_HSP90-amp-plots.png) | ![Cg_HSP90 melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Cg_HSP90-melt-plots.png) | | Cg_VIPERIN | ![Cg_VIPERIN amplification plot](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Cg_VIPERIN-amp-plots.png) | ![Cg_VIPERIN melt curve](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-19%2011-54-14_CFX96-Cg_VIPERIN-melt-plots.png) |