Hi Jitka,

Overall, the telomere qPCR is most of the most difficult pcr assays i have worked with to optimize. We eventaully settled on a commercial kit that works really well. This kit includes a human gDNA reference sample with a known telomere Kb length, so you can do absolute quantification. Further, the standards are made from human gDNA so the reaction kinetcis between your samples and the standards will be very similar. This is the absolute telomere pcr quantification kit by ScienceCell. It saved us a lot of time and headaches in the end. If you want to continue with your current approach read below.

I assume you are using the Cawthon method for telomere qPCR? This is a good starting place. I would recommend avoiding the O'Callaghan method as it used a synthetic nucleotide standard, which does not have same reaction kinetics as isolated gDNA.

As Alireza Mordadi stated, you need to increase the number of cycles. 25 is too low; you will not reach the plateu with this assay at 25. I would recommend 32 to 35 cycles because of the single copy gene (SCG) will amplify much later than the telomere. I would also recommend measuring each sample in triplicate (both telomere and SCG). This is because the SCG can amplify very late, and the closer you get to Ct of 30, the more variation between replicates there is. With triplicates you can either discard the outlier well, and/or increase precision if you get average Ct from three wells.

For our telomere qPCR, we use between 1 to 5 ng of gDNA per reaction well. This is plenty, no need to go higher.

"The secong problem is, that I have lower amount of telomere product than product of single copy gene (Ct about 22). When I take a look on melting courves, I really have only one product..."

I see from the software you have selected fluorophore analysis (top right of second image). You should analyse by target. The SCG and telomere reactions do not have same reaction kinetics, you do not want the software to average kinetics and calcualtions for both targets together. I cannot tell from the image you have provided much about melting curve. Can you provide an image of each targets melting curves? Also, can you clarify what the Ct of the telomere AND SCG is? You mention one is 22, but it is difficult to see from picture which one this is.

Second, (see point 7 below), a common SCG used in telomere qPCR has many pseudogenes. If your primer is also binding to the pseudogenes, you might get many genes amplified for the SCG, which could make Ct of SCG lower than Ct of telomere.

Some other tips for telomere qPCR.

1. Singleplex worked best for us. However, be aware that, depending on your master mix and pcr machine, you need completely different melting and annealing temepratures for the telomere primers compared to SCG primers. If this is the case, it is possible you might need to have two plates, one for telomere and one for SCG.

2. The telomere primers will form a primer-dimer (limitation with telomere qPCR), and will amplify sometimes within 8 cycles of you telomere samples. You MUST include telomere no template wells (NTC: wells with no gDNA, but everything else), to show that the primer dimer has different melt curve charactersitics. This way you can demonstrate your gDNA telomere samples have different melting curves than NTC telomere, so your gDNA telomere Ct values are not a false postiive from primer-dimer.

3. Everything in triplicate, at a minimum. This assay is very sensitive.

4. Always use same well postition for standards, controls, NTCs, etc, on different plates. Again, because this assay is so sensitive you have to control for very small temperature differences in your PCR machine heating block. We had a 0.1C different across our block at one point, which could change sample Ct if standards were in different well positions on different plates. We reduced this issue by using same wells positons on every plate (Improving qPCR telomere length assays: Controlling for well position effects increases statistical power, 2015).

5. Primer concentrations are unsual in this assay. Many groups report for example, that the telomere forward primer is 300 nM, while telomere reverse primer is at 900 nM. This really depends on your master mix and PCR machine. You have to try a very broad matrix of primer concentrations, with more than one individuals gDNA! The reaction efficiency has to work well for several individuals gDNA.

6. You need really clean gDNA. Test A260/A280 absorbance (protein contamination) and also A260/A230 (salts). Because there are so few copies of the SCG, contamination might affect this assay.

7. Many groups that do telomere qPCR with Cawthon method use the SCG RPLP0/36B4. This is a bad SCG! It has some pseudogenes that the common primers for 36B4 can bind to. Check using Reverse-Primer Blast that your primers only target 36B4 and not the pseudogenes, but as our group saw the common primers for 36B4 have a pretty close binding energy to pseudogenes as well as the actual gene. A better SCG target is albumin, which has far less/no pseudogenes.

8. Always run stanadrd curves on each plate for both telomere and SCG. Because the SCG at the last dilution may amplify really late (30+) it is possible that your standard curve reaction efficiency will change by +/- 5%. You really should correct your Ct equation for the reaction efficeiny per plate. This is the Pfaffl efficiency correction method for relative and absolute quantification in PCR.

9. gDNA Isolation methods effect telomere length assesment. Always isolate everything using same kit!

I didn't understand you completely.

Why you had only 25 cycles?

There is no need more DNA. Raise your cycles to reach platue.

There is two different genes that you expect two different melt curves?

Melting curve is based on sequence and length of product so it's possible you have the same curve even for two different Gene's. Change the primers.

Regards.

Hi Jitka Smetanova,

If I were you I would not increase DNA concentration for the reason that: scg is single copy, while your telomere repeats should be at least more copies, therefore first play till you sure have amplification with telomere primer (alone). another important point you should look is primer concentration for telomere.

Goodluck

Hi,

I agree with both. In addition I would run a temp gradient to see if you have the optimal cylcing conditions.

An opposing to using more DNA I would rather consider to use less to be able to resolute multi copies better

Hi Jitka,

Overall, the telomere qPCR is most of the most difficult pcr assays i have worked with to optimize. We eventaully settled on a commercial kit that works really well. This kit includes a human gDNA reference sample with a known telomere Kb length, so you can do absolute quantification. Further, the standards are made from human gDNA so the reaction kinetcis between your samples and the standards will be very similar. This is the absolute telomere pcr quantification kit by ScienceCell. It saved us a lot of time and headaches in the end. If you want to continue with your current approach read below.

I assume you are using the Cawthon method for telomere qPCR? This is a good starting place. I would recommend avoiding the O'Callaghan method as it used a synthetic nucleotide standard, which does not have same reaction kinetics as isolated gDNA.

As Alireza Mordadi stated, you need to increase the number of cycles. 25 is too low; you will not reach the plateu with this assay at 25. I would recommend 32 to 35 cycles because of the single copy gene (SCG) will amplify much later than the telomere. I would also recommend measuring each sample in triplicate (both telomere and SCG). This is because the SCG can amplify very late, and the closer you get to Ct of 30, the more variation between replicates there is. With triplicates you can either discard the outlier well, and/or increase precision if you get average Ct from three wells.

For our telomere qPCR, we use between 1 to 5 ng of gDNA per reaction well. This is plenty, no need to go higher.

"The secong problem is, that I have lower amount of telomere product than product of single copy gene (Ct about 22). When I take a look on melting courves, I really have only one product..."

I see from the software you have selected fluorophore analysis (top right of second image). You should analyse by target. The SCG and telomere reactions do not have same reaction kinetics, you do not want the software to average kinetics and calcualtions for both targets together. I cannot tell from the image you have provided much about melting curve. Can you provide an image of each targets melting curves? Also, can you clarify what the Ct of the telomere AND SCG is? You mention one is 22, but it is difficult to see from picture which one this is.

Second, (see point 7 below), a common SCG used in telomere qPCR has many pseudogenes. If your primer is also binding to the pseudogenes, you might get many genes amplified for the SCG, which could make Ct of SCG lower than Ct of telomere.

Some other tips for telomere qPCR.

1. Singleplex worked best for us. However, be aware that, depending on your master mix and pcr machine, you need completely different melting and annealing temepratures for the telomere primers compared to SCG primers. If this is the case, it is possible you might need to have two plates, one for telomere and one for SCG.

2. The telomere primers will form a primer-dimer (limitation with telomere qPCR), and will amplify sometimes within 8 cycles of you telomere samples. You MUST include telomere no template wells (NTC: wells with no gDNA, but everything else), to show that the primer dimer has different melt curve charactersitics. This way you can demonstrate your gDNA telomere samples have different melting curves than NTC telomere, so your gDNA telomere Ct values are not a false postiive from primer-dimer.

3. Everything in triplicate, at a minimum. This assay is very sensitive.

4. Always use same well postition for standards, controls, NTCs, etc, on different plates. Again, because this assay is so sensitive you have to control for very small temperature differences in your PCR machine heating block. We had a 0.1C different across our block at one point, which could change sample Ct if standards were in different well positions on different plates. We reduced this issue by using same wells positons on every plate (Improving qPCR telomere length assays: Controlling for well position effects increases statistical power, 2015).

5. Primer concentrations are unsual in this assay. Many groups report for example, that the telomere forward primer is 300 nM, while telomere reverse primer is at 900 nM. This really depends on your master mix and PCR machine. You have to try a very broad matrix of primer concentrations, with more than one individuals gDNA! The reaction efficiency has to work well for several individuals gDNA.

6. You need really clean gDNA. Test A260/A280 absorbance (protein contamination) and also A260/A230 (salts). Because there are so few copies of the SCG, contamination might affect this assay.

7. Many groups that do telomere qPCR with Cawthon method use the SCG RPLP0/36B4. This is a bad SCG! It has some pseudogenes that the common primers for 36B4 can bind to. Check using Reverse-Primer Blast that your primers only target 36B4 and not the pseudogenes, but as our group saw the common primers for 36B4 have a pretty close binding energy to pseudogenes as well as the actual gene. A better SCG target is albumin, which has far less/no pseudogenes.

8. Always run stanadrd curves on each plate for both telomere and SCG. Because the SCG at the last dilution may amplify really late (30+) it is possible that your standard curve reaction efficiency will change by +/- 5%. You really should correct your Ct equation for the reaction efficeiny per plate. This is the Pfaffl efficiency correction method for relative and absolute quantification in PCR.

9. gDNA Isolation methods effect telomere length assesment. Always isolate everything using same kit!

Hi,

thanks everybody for answers. Yesterday, I increased the number of cycles. I set up 33 cycles at all. Yes Mr. Frankl, you are right. At the begining, I followed Cawthon method. But the telomere primers did not work to me, the amplification plot had linear shape. The second problem was, that Cawthon multiplex method is based on two different temperature reading. But in my case, the machine read only first one (I saw changes in courves), during second one nothing happened. Because of this reasson I used primers for telomere from Hubacek et al. (2012) and separated telomere and scg (I use albumin). With mentioned primers, the PCR courves look much better. You were right, when I rised up the cycles, I got plateu phase. Now telomere looks almost well I think (pictures for telomere attached). Now, the concentration of primers for telomere is 900 nm for F and 450 nm for R.

Pictures, whitch I posted two days ago, have only products of telomere. Now, I dont optimize scg (albumin), because it runs well at the moment. When I am at work, I will send the picture of albumin product as well.

Software set up: When I set up the method on the cycler (CFX96), I set up the program, and I use analyzation SYBR/FAM only. Where can I set up analyzation acording target?

Thanks a lot,

Nice day all.

Hi

During analysis you can select in the upper left part analysis by target or fluorophor but you need to mark them.

In addition you can add more reads to enable readout at different temperatures if needed

Sven