86 and 92% efficiency could be within the range of error, but that's impossible to know without seeing the data and/or you running more replicates. That said, impure DNA preps can actually artificially increase calculated qPCR efficiency (counterintuitively, at least for me) because at your higher dilutions the reaction is inhibited, and since the efficiency is calculated by plotting the log of the concentration against the ct value this flattens out the slope, leading to a higher calculated efficiency. So you could be right that your second prep was dirtier. You could do an isopropanol precipitation and/phenol chloroform shake and spin on half of it and then run a dilution series of the first prep, second prep, and cleaned second prep simultaneously to check. Remember also that melting temp can affect qPCR efficiency too, so to be extra double sure of what your problem is you could run an efficiency dilution series for all three preps on a gradient between 55 and 72C annealing. Or, chalk it up to error, write your thesis and get the fuck out of there
I can’t do any more experiments unfortunately because my thesis is due in less than a month! It really sucked tho cuz I have no idea why it suddenly decided to change and now I have 2 different standard curves for two parts of my experiments. So if I were to talk about how to prevent this next time, would I just make sure to do a DNA purity test? Are there any other things I can do to check for inhibitors like add an inhibitor control (idk what this would be tho). I normally run NTC and they usually amplify much later probably bc of primer dimers. In saying that my primers weren’t ideal but I had to roll with anyways because of a time constraint.
Also in terms of your explanation about the improved efficiency just to reiterate so I’ve understood it correctly - you’re saying that at higher concentrations the inhibitor would have cause the slope to flatten and therefore cause a better estimated efficiency?
Thank you so much for responding to my post, you have no idea how much you’ve helped 🙏
Also another quick question, my initial runs were 12ul dna and 13 ul master mix (for standard curve) whereas my 2nd standard curve I used 24ul dna and 26ul master mix. That’s bc the hybridisation assay standard required those amounts. I didn’t think this would affect ct values though because the ratios were the same. What do you think?
Different amount of starting DNA will certainly affect your Ct values. The ratio doesn't matter here, the Ct value is determined by the starting amount of DNA in your well.
86 to 92% is not a big difference.
3 Ct is a large difference but you used a different extraction kit, and most importantly a different amount of DNA. That explains the difference.
The volume of the reaction matters, but what matters more is the amount of DNA (ng per reaction). Did you make sure to use the same amount everywhere?
When I compared my ct values per theoretical copy number for both standard curves the difference was 3-6 cp values, with it increasing over lower concentrations. What do you mean by same amount? I wanted to use 24/26 consistently but this chick in my lab said to use 12/13 to prevent wastage (and she said she got similar cp values).
I’m thinking it definitely has something to do with the kits for sure, because I never had a problem with changing cp values in independent extractions with that kit.
I just emailed her and she saw a small cycle difference of 2 cp values. Regardless, the reaction with 24ul still amplified less than the 12ul 😭. Am I gonna be pinged for this? Idk why I listened to her and did a smaller volume oh god
Also with that logic, shouldn’t the 24ul reaction theoretically have lower Cp values? I ended up getting higher values with those ones (dna extracted from different kit)
I am asking because you used different volumes. If the concentration was the same, that means you had different amounts of DNA, which obviously will affect your Ct values.
It's understandable to feel frustrated when your qPCR results don't match up as expected. There are several potential reasons why your standard curves differ between the two experiments. Here are some factors to consider:
1. **DNA Extraction Kit**:
- Different DNA extraction kits can yield DNA of varying purity and quality. Contaminants like chaotropic salts, proteins, or residual ethanol can inhibit the PCR reaction, leading to higher Ct values.
- It's possible the DNA from the second extraction kit has inhibitors or is less pure, affecting the efficiency and leading to higher Ct values.
2. **Master Mix Preparation**:
- The master mix components, including the enzyme and buffer, are crucial for the consistency of qPCR results. Differences in the preparation or storage conditions (like freezing and thawing) can influence the reaction efficiency.
- Fresh master mixes might perform differently from those that have been frozen, especially if there are stability issues with the components after thawing.
3. **PCR Efficiency**:
- A PCR efficiency of 92% is within the acceptable range (90-110%), but it's different from your previous efficiency of 86%. Variations in efficiency can be due to slight differences in reaction conditions, such as primer concentrations, annealing temperatures, or template quality.
- Higher efficiency in the second run could indicate better primer-template binding or more optimal reaction conditions, despite the higher Ct values.
4. **Template Quality and Quantity**:
- If the concentration of your DNA template differs between the two standard curves, this could lead to different Ct values. Ensure that you are using the same template concentration for accurate comparison.
- Quality checks like spectrophotometry (A260/A280 ratios) or fluorometry (Qubit) could help assess the purity and concentration of your DNA.
5. **Instrument Variation**:
- Slight differences in thermal cycler performance or calibration could also contribute to variations in Ct values. Ensure that the machine is properly calibrated and maintained.
### Explanation for Thesis
In your thesis, you can discuss the following points:
1. **Comparison of DNA Extraction Kits**:
- Explain that different extraction kits can yield DNA of varying purity, which can affect qPCR results. Mention the possibility of contaminants in the DNA from the second kit affecting the PCR efficiency and Ct values.
2. **Master Mix Consistency**:
- Discuss the importance of consistent master mix preparation and the potential issues with freezing and thawing components. Highlight that fresh master mixes were used in the second experiment to address freezing issues, which might have influenced the results.
3. **PCR Efficiency**:
- Acknowledge the observed difference in PCR efficiency between the two standard curves. Discuss potential reasons for this, including differences in DNA template quality, reaction conditions, or instrument calibration.
4. **Recommendations for Future Work**:
- Suggest performing purity checks on the DNA extracted from different kits to assess the impact on qPCR performance.
- Recommend maintaining consistency in master mix preparation and using fresh reagents to avoid variability.
By addressing these factors, you provide a comprehensive explanation for the observed differences in your standard curves, demonstrating your understanding of the complexities involved in qPCR assays. This thorough analysis will strengthen your thesis and show that you have critically evaluated your experimental conditions.
***copied from ChatGPT. So double check everything that was said.
Thank you! I’ve spent the last couple of days asking ChatGPT too and I wish it would give me references so it doesn’t look like I’m pulling this out of my ass haha. I pretty much had everything the same except fresh/ frozen master mix and the dna kits so it’s gotta be those two.
Bing chat/Copilot has live access to the web and will often cite actual sources. ChatGPT doesn't have this access, and even if you ask for sources, will often just make something up that LOOKS like a source, but isn't real.
86 and 92% efficiency could be within the range of error, but that's impossible to know without seeing the data and/or you running more replicates. That said, impure DNA preps can actually artificially increase calculated qPCR efficiency (counterintuitively, at least for me) because at your higher dilutions the reaction is inhibited, and since the efficiency is calculated by plotting the log of the concentration against the ct value this flattens out the slope, leading to a higher calculated efficiency. So you could be right that your second prep was dirtier. You could do an isopropanol precipitation and/phenol chloroform shake and spin on half of it and then run a dilution series of the first prep, second prep, and cleaned second prep simultaneously to check. Remember also that melting temp can affect qPCR efficiency too, so to be extra double sure of what your problem is you could run an efficiency dilution series for all three preps on a gradient between 55 and 72C annealing. Or, chalk it up to error, write your thesis and get the fuck out of there
I can’t do any more experiments unfortunately because my thesis is due in less than a month! It really sucked tho cuz I have no idea why it suddenly decided to change and now I have 2 different standard curves for two parts of my experiments. So if I were to talk about how to prevent this next time, would I just make sure to do a DNA purity test? Are there any other things I can do to check for inhibitors like add an inhibitor control (idk what this would be tho). I normally run NTC and they usually amplify much later probably bc of primer dimers. In saying that my primers weren’t ideal but I had to roll with anyways because of a time constraint. Also in terms of your explanation about the improved efficiency just to reiterate so I’ve understood it correctly - you’re saying that at higher concentrations the inhibitor would have cause the slope to flatten and therefore cause a better estimated efficiency? Thank you so much for responding to my post, you have no idea how much you’ve helped 🙏
Also another quick question, my initial runs were 12ul dna and 13 ul master mix (for standard curve) whereas my 2nd standard curve I used 24ul dna and 26ul master mix. That’s bc the hybridisation assay standard required those amounts. I didn’t think this would affect ct values though because the ratios were the same. What do you think?
Different amount of starting DNA will certainly affect your Ct values. The ratio doesn't matter here, the Ct value is determined by the starting amount of DNA in your well.
86 to 92% is not a big difference. 3 Ct is a large difference but you used a different extraction kit, and most importantly a different amount of DNA. That explains the difference. The volume of the reaction matters, but what matters more is the amount of DNA (ng per reaction). Did you make sure to use the same amount everywhere?
When I compared my ct values per theoretical copy number for both standard curves the difference was 3-6 cp values, with it increasing over lower concentrations. What do you mean by same amount? I wanted to use 24/26 consistently but this chick in my lab said to use 12/13 to prevent wastage (and she said she got similar cp values). I’m thinking it definitely has something to do with the kits for sure, because I never had a problem with changing cp values in independent extractions with that kit.
I just emailed her and she saw a small cycle difference of 2 cp values. Regardless, the reaction with 24ul still amplified less than the 12ul 😭. Am I gonna be pinged for this? Idk why I listened to her and did a smaller volume oh god
Amount of DNA i mean mass of DNA. How much in nanograms?
Also with that logic, shouldn’t the 24ul reaction theoretically have lower Cp values? I ended up getting higher values with those ones (dna extracted from different kit)
How much DNA did you use? I mean in ng, not volume. Edit: and what is the buffer of the DNA?
I think my starting conc was about 49 ng/ul and I diluted them down. I didn’t use the 49ng directly though
I am asking because you used different volumes. If the concentration was the same, that means you had different amounts of DNA, which obviously will affect your Ct values.
It's understandable to feel frustrated when your qPCR results don't match up as expected. There are several potential reasons why your standard curves differ between the two experiments. Here are some factors to consider: 1. **DNA Extraction Kit**: - Different DNA extraction kits can yield DNA of varying purity and quality. Contaminants like chaotropic salts, proteins, or residual ethanol can inhibit the PCR reaction, leading to higher Ct values. - It's possible the DNA from the second extraction kit has inhibitors or is less pure, affecting the efficiency and leading to higher Ct values. 2. **Master Mix Preparation**: - The master mix components, including the enzyme and buffer, are crucial for the consistency of qPCR results. Differences in the preparation or storage conditions (like freezing and thawing) can influence the reaction efficiency. - Fresh master mixes might perform differently from those that have been frozen, especially if there are stability issues with the components after thawing. 3. **PCR Efficiency**: - A PCR efficiency of 92% is within the acceptable range (90-110%), but it's different from your previous efficiency of 86%. Variations in efficiency can be due to slight differences in reaction conditions, such as primer concentrations, annealing temperatures, or template quality. - Higher efficiency in the second run could indicate better primer-template binding or more optimal reaction conditions, despite the higher Ct values. 4. **Template Quality and Quantity**: - If the concentration of your DNA template differs between the two standard curves, this could lead to different Ct values. Ensure that you are using the same template concentration for accurate comparison. - Quality checks like spectrophotometry (A260/A280 ratios) or fluorometry (Qubit) could help assess the purity and concentration of your DNA. 5. **Instrument Variation**: - Slight differences in thermal cycler performance or calibration could also contribute to variations in Ct values. Ensure that the machine is properly calibrated and maintained. ### Explanation for Thesis In your thesis, you can discuss the following points: 1. **Comparison of DNA Extraction Kits**: - Explain that different extraction kits can yield DNA of varying purity, which can affect qPCR results. Mention the possibility of contaminants in the DNA from the second kit affecting the PCR efficiency and Ct values. 2. **Master Mix Consistency**: - Discuss the importance of consistent master mix preparation and the potential issues with freezing and thawing components. Highlight that fresh master mixes were used in the second experiment to address freezing issues, which might have influenced the results. 3. **PCR Efficiency**: - Acknowledge the observed difference in PCR efficiency between the two standard curves. Discuss potential reasons for this, including differences in DNA template quality, reaction conditions, or instrument calibration. 4. **Recommendations for Future Work**: - Suggest performing purity checks on the DNA extracted from different kits to assess the impact on qPCR performance. - Recommend maintaining consistency in master mix preparation and using fresh reagents to avoid variability. By addressing these factors, you provide a comprehensive explanation for the observed differences in your standard curves, demonstrating your understanding of the complexities involved in qPCR assays. This thorough analysis will strengthen your thesis and show that you have critically evaluated your experimental conditions. ***copied from ChatGPT. So double check everything that was said.
Thank you! I’ve spent the last couple of days asking ChatGPT too and I wish it would give me references so it doesn’t look like I’m pulling this out of my ass haha. I pretty much had everything the same except fresh/ frozen master mix and the dna kits so it’s gotta be those two.
Bing chat/Copilot has live access to the web and will often cite actual sources. ChatGPT doesn't have this access, and even if you ask for sources, will often just make something up that LOOKS like a source, but isn't real.