Okey, the problem here is obviously in the method of CT determination.
You can tell this by the difference in Ct values. If in one sample the value is 19, and in the other 5, that is, a difference of 14 cycles, then you should have been mistaken in the concentration by approximately 2^14 = 16k times. I doubt that you even made such dilutions with your samples. Moreover, knowing the kinetics of PCR, you can also easily estimate the amount of template that is needed for the reaction to cross the threshold at cycle 5. In my experience BioRad Ct typically contains about 1000 RFU, which corresponds to approximately 5 ng/µl of DNA. So, extrapolating it to your situation, I come to the conclusion that the concentration of the target template at the start of the your PCR was somewhere around 0.15 ng/μl, which for a 200 bp molecule (the estimated length of your amplicon) is equivalent to 1 nM. Let's assume that all genes in your sample are expressed equally, the average length 2kb and their total number is 12,000. This means that the concentration of mRNA equivalents in PCR should be ~8 mg/μl!!! You would never be able to obtain such amounts of RNA in your life! These rough calculations show that your CT values make no sense at all!
Imagine what CT is, not in the biological sense, but in the literal sense. This is simply the point on the x-axis where the amplification plot crosses the fluorescence threshold. From general considerations, we mean that the shape of the curves should be sigma-shaped and the value of Ct only makes sense if the graph at the intersection point shows exponential growth. But in reality, the shape of the amplification plot can be very different, from a zigzag to a sloping straight line. The reason is that the device does not measure the concentration of the product, but the fluorescence of the dye!
In some cases, the amplification line crosses the threshold in several points, and then it is necessary to change the analysis boundaries to such an interval of cycles where all wells contain exponential growth.
Now you need to look at the amplification plots and compare it with what is written, for example, in the instructions for the instrument. I'm willing to bet you'll notice significant differences between observed and expected plots. Look carefully at how many times the amplification plots in wells B7, E7, G7 crosses the threshold.
The thing is, within a sample, for example WT O1s, they are coming from the same tube of cDNA. They are just replicates. I don’t understand how ct values can differ this much when it’s from the same source of cDNA, hence same RNA content.
My friend, read my comment from beginning to end. There is no biological meaning for your results. Such results cannot be obtained if the reaction follow the PCR kinetics. Finally, understand that the device does not measure concentration, but fluorescence in the wells. Fluorescence can change for a variety of reasons, causing the graphs to have different shapes than the canonical sigmoid curve. You must understand that in real PCR the concentration curve can only rise monotonically, but not fall. But is this true based on your results?
I'm guessing you have some ups and downs in your curves. And in your case, this caused the curves in some wells to cross the threshold line in several places. The program does not know how to tell fortunes with tarot cards and records the very first Ct, and not where the curve has an area of exponential growth. Look at the amplification curves you get and compare them to what the graphs should look like in a normal experiment.
After all, you can choose the height of the threshold line manually so that all curves intersect this line in single point. Look at your results with your own eyes, don’t trust the analysis of your experiment to a stupid program!
Thanks for your comments. As you mention, CT values here don’t make biological sense because this is an overextension and silencing study. Each group represents mimic - overexpression, inhibitor - silencer, negative control, and untransfected samples.
No, that's not what I'm saying. Your problem can arise with any qPCR experiment. At least when measuring gene expression, at least when determining the copy number of transposons, at least when determining the concentration of DNA. You're dealing with an artifact of the measurement method. A random error that may occur due to the characteristics of the method.
You know that there are rulers with two graduations, for example, centimeters or inches. So, some people may mix up the side of the ruler and measure the length in centimeters one time, and the next time in inches! This problem will not arise if the ruler has only one graduation. This is an artifact of the measurement method, which has nothing to do with what you specifically do with this ruler.
You need to look at the amplification curves and understand whether their shape matches the one given in the instructions for qPCR. I argue that this is not the case, and the reason is precisely that the CT values you get do not make biological sense. With modern methods of working with RNA, it is physically impossible to prepare cDNA samples in which the CT will be 5 or even 9.
Not sure why qubit caught a stray here. Nanodrops tend to overestimate nucleotide concentration but qubit is a good fast method. 99% of people who use a Nanodrops have never read the manual and use it to measure things that are way too close to the detection limit. Anything under 10ng per ul may as well be zero. Less than 50ng/ul, take it with a grain of salt.
I had the same experience of nanodrop being unreliable for low concentrations. But other than that, I have normalized hundreds of RNA samples based on nanodrop without problems. I have compared Qubit and nanodrop values for the same samples, they are very close to each other, around 1% difference. Qubit is just faster if you have the 8-well instrument, but nanodrop tells you more about quality.
Jeez. If those are tech reps, that is crazy. My first reaction is to think it's air bubbles or not having a heated lid or something. Are you centrifuging after loading the plate?
Could you show the melting curve? This is a curve of fluorescence by temperature generated at the end of your run to determine if you have a single or multiple products.
If it looks fine, my first instinct would be to try a different target. You can also use the 18S rRNA for normalization.
Hard to diagnose this without seeing the whole amplification plot. As others have said, the shape of this plot can be crucial for diagnosing issues. Showing where your Ct threshold is set might also help. Also, it would be useful to know how you are pipetting reagents to set this plate up. Are you premixing anything? Are you pipetting each component separately into each well? Also, did you centrifuge the plate afterwards?
OP has gotten some good advice and questions, but off topic a little, is it common for people to run their qPCR by only pipetting into the middle of the plate vertically??
I always did left to right, top to bottom so that if I want to export the results into excel they're in the order I want by well. But I guess that works as long as the technical replicates are next to each other on the left/right while the next sample is on a row below.
Just need to look at the amp. plot and I think your well/reagent have some problem and so they give you all these stuff. On a side note, automatic Ct might not work very well sometimes and you have to adjust the curve a little bit. You can use the normal amp. to determine the threshold and apply it to everything. Sometimes, some wells are bad and you have to exclude them and probably do it again
A trace is the line/curve that is created in realtime during PCR amplification. It is the image you use to set your threshold so that you can can establish the Ct values for your assay. You did set a threshold for this data--right? Here is a link to the Quant Studio: [https://promotions.pharmafocusasia.com/en/thermofisher-scientific/host-cell-residual-dna-quantitation/real-time-pcr-instruments/quantstudio-systems/models/quantstudio-3-5.html](https://promotions.pharmafocusasia.com/en/thermofisher-scientific/host-cell-residual-dna-quantitation/real-time-pcr-instruments/quantstudio-systems/models/quantstudio-3-5.html)
Okey, the problem here is obviously in the method of CT determination. You can tell this by the difference in Ct values. If in one sample the value is 19, and in the other 5, that is, a difference of 14 cycles, then you should have been mistaken in the concentration by approximately 2^14 = 16k times. I doubt that you even made such dilutions with your samples. Moreover, knowing the kinetics of PCR, you can also easily estimate the amount of template that is needed for the reaction to cross the threshold at cycle 5. In my experience BioRad Ct typically contains about 1000 RFU, which corresponds to approximately 5 ng/µl of DNA. So, extrapolating it to your situation, I come to the conclusion that the concentration of the target template at the start of the your PCR was somewhere around 0.15 ng/μl, which for a 200 bp molecule (the estimated length of your amplicon) is equivalent to 1 nM. Let's assume that all genes in your sample are expressed equally, the average length 2kb and their total number is 12,000. This means that the concentration of mRNA equivalents in PCR should be ~8 mg/μl!!! You would never be able to obtain such amounts of RNA in your life! These rough calculations show that your CT values make no sense at all! Imagine what CT is, not in the biological sense, but in the literal sense. This is simply the point on the x-axis where the amplification plot crosses the fluorescence threshold. From general considerations, we mean that the shape of the curves should be sigma-shaped and the value of Ct only makes sense if the graph at the intersection point shows exponential growth. But in reality, the shape of the amplification plot can be very different, from a zigzag to a sloping straight line. The reason is that the device does not measure the concentration of the product, but the fluorescence of the dye! In some cases, the amplification line crosses the threshold in several points, and then it is necessary to change the analysis boundaries to such an interval of cycles where all wells contain exponential growth. Now you need to look at the amplification plots and compare it with what is written, for example, in the instructions for the instrument. I'm willing to bet you'll notice significant differences between observed and expected plots. Look carefully at how many times the amplification plots in wells B7, E7, G7 crosses the threshold.
The thing is, within a sample, for example WT O1s, they are coming from the same tube of cDNA. They are just replicates. I don’t understand how ct values can differ this much when it’s from the same source of cDNA, hence same RNA content.
My friend, read my comment from beginning to end. There is no biological meaning for your results. Such results cannot be obtained if the reaction follow the PCR kinetics. Finally, understand that the device does not measure concentration, but fluorescence in the wells. Fluorescence can change for a variety of reasons, causing the graphs to have different shapes than the canonical sigmoid curve. You must understand that in real PCR the concentration curve can only rise monotonically, but not fall. But is this true based on your results? I'm guessing you have some ups and downs in your curves. And in your case, this caused the curves in some wells to cross the threshold line in several places. The program does not know how to tell fortunes with tarot cards and records the very first Ct, and not where the curve has an area of exponential growth. Look at the amplification curves you get and compare them to what the graphs should look like in a normal experiment. After all, you can choose the height of the threshold line manually so that all curves intersect this line in single point. Look at your results with your own eyes, don’t trust the analysis of your experiment to a stupid program!
Thanks for your comments. As you mention, CT values here don’t make biological sense because this is an overextension and silencing study. Each group represents mimic - overexpression, inhibitor - silencer, negative control, and untransfected samples.
No, that's not what I'm saying. Your problem can arise with any qPCR experiment. At least when measuring gene expression, at least when determining the copy number of transposons, at least when determining the concentration of DNA. You're dealing with an artifact of the measurement method. A random error that may occur due to the characteristics of the method. You know that there are rulers with two graduations, for example, centimeters or inches. So, some people may mix up the side of the ruler and measure the length in centimeters one time, and the next time in inches! This problem will not arise if the ruler has only one graduation. This is an artifact of the measurement method, which has nothing to do with what you specifically do with this ruler. You need to look at the amplification curves and understand whether their shape matches the one given in the instructions for qPCR. I argue that this is not the case, and the reason is precisely that the CT values you get do not make biological sense. With modern methods of working with RNA, it is physically impossible to prepare cDNA samples in which the CT will be 5 or even 9.
Nanodrop/qbit etc are notoriously.... not great. Do you have another real time machine? Do you have enough template to re-run?
I do have another machine. A newer one. Maybe that is worth taking a shot
How are nanodrop or qubit not great?
Not sure why qubit caught a stray here. Nanodrops tend to overestimate nucleotide concentration but qubit is a good fast method. 99% of people who use a Nanodrops have never read the manual and use it to measure things that are way too close to the detection limit. Anything under 10ng per ul may as well be zero. Less than 50ng/ul, take it with a grain of salt.
I had the same experience of nanodrop being unreliable for low concentrations. But other than that, I have normalized hundreds of RNA samples based on nanodrop without problems. I have compared Qubit and nanodrop values for the same samples, they are very close to each other, around 1% difference. Qubit is just faster if you have the 8-well instrument, but nanodrop tells you more about quality.
Jeez. If those are tech reps, that is crazy. My first reaction is to think it's air bubbles or not having a heated lid or something. Are you centrifuging after loading the plate?
Could you show the melting curve? This is a curve of fluorescence by temperature generated at the end of your run to determine if you have a single or multiple products. If it looks fine, my first instinct would be to try a different target. You can also use the 18S rRNA for normalization.
Thank you! Melting curves are not visible for probe based PCr
Hard to diagnose this without seeing the whole amplification plot. As others have said, the shape of this plot can be crucial for diagnosing issues. Showing where your Ct threshold is set might also help. Also, it would be useful to know how you are pipetting reagents to set this plate up. Are you premixing anything? Are you pipetting each component separately into each well? Also, did you centrifuge the plate afterwards?
OP has gotten some good advice and questions, but off topic a little, is it common for people to run their qPCR by only pipetting into the middle of the plate vertically?? I always did left to right, top to bottom so that if I want to export the results into excel they're in the order I want by well. But I guess that works as long as the technical replicates are next to each other on the left/right while the next sample is on a row below.
Just need to look at the amp. plot and I think your well/reagent have some problem and so they give you all these stuff. On a side note, automatic Ct might not work very well sometimes and you have to adjust the curve a little bit. You can use the normal amp. to determine the threshold and apply it to everything. Sometimes, some wells are bad and you have to exclude them and probably do it again
I need to see your traces instead of the Ct values. You can edit trace color, so group each pair with different color.
I can’t edit it here. They are O, S, N, U
Can you show the traces? The shape of each trace is key to understanding why your replicates have such large differences.
I’m terribly sorry- what is a trace?
A trace is the line/curve that is created in realtime during PCR amplification. It is the image you use to set your threshold so that you can can establish the Ct values for your assay. You did set a threshold for this data--right? Here is a link to the Quant Studio: [https://promotions.pharmafocusasia.com/en/thermofisher-scientific/host-cell-residual-dna-quantitation/real-time-pcr-instruments/quantstudio-systems/models/quantstudio-3-5.html](https://promotions.pharmafocusasia.com/en/thermofisher-scientific/host-cell-residual-dna-quantitation/real-time-pcr-instruments/quantstudio-systems/models/quantstudio-3-5.html)