Hi Goutham

you did a real job in constructing a kind protocol to analyze copy number of your target. What I would add is a point on the primers themselves, the length of the amplicons and the way to analyze. there are lot of characteristics that primers need to have or not (% GC not too high, a Tm around 55-60 and a Tm difference between primers under 2°c, G or C at 3' and 5', avoid primers interactions...), the amplicons length must be between 100 and 150, and you must follow the ddCT protocol to analyse the results. Also checking the amplification by in silico PCR at the UCSC or NCBI website should be best.

fred

First of all, target gene and software used for oligos development. Next: as Frederic mentioned, avoid G or C on 5' end of the probe (especially G). Cut your plasmid to obtain linear DNA. Be sure if there is around 5ºC difference between Tm of your primers and probe (e.g. 60 for primers, 65 for probe). Check your method with external standard (e.g. DNA or cDNA from virus cultivated in cell culture). Manipulate with annealing temperature: try 53, 55, 57 and 60 degrees of Celsius, check how it influences the method's ability to detect low-copy DNA. Work rather with 10e3 - 10e4 plasmid copies than 10e6. It will show repeteability of the method. If there are differences between repetitions, it means that something is wrong with reaction kinetics.

Hello Maciej Przybylski and Fredrick

I here share all the parameters of primers and probes, we did standardize the annealing temperatures required for the real time PCR accordingly (via gradient pcr)

Yes I shall consider using 10e3/10e4 for next time.

The only issue I can view wrt primers is the GC content of Forward and Reverse, I did try using different annealing time but it didnt give any significant shift in Ct values.

Hi Goutham,

as we can't see in the picture, did you ensure that Tm difference between each primers and each couple of primers do not exceed 2°c. GC% is not really different, but Tm is also depending on the length of primers. you can play with that to adjust the Tm in a delta of 2°c, always respecting the rules of G and C at 3 and 54 ends, and avoiding dimerisations and interactions (but as I can see the deltaG is under 5, good it is). the ideal way is to take for this match players with the same qualities and defaults...

fred

Yes Fred the Tm difference between F and R is not more than 2 C, and the 3` end of both the primers do have G and C the GC clamp number in the image represents them,

Now I do have strong feeling that as there are no primer dimers and very minimal cross dimers with probe the designed primers and probes should give better amplification and signal, I did try using the primers alone in SyBr green reaction and it did gave better amplification plots compared to probe based for lower copy number dilutions,

I might have to try the same reaction with different brand master mix which may give me a better picture.

what do u think ?

Master mix shouldn't be a problem. Of course, it should be obtained from reliable source. But if people work with it worldwide and it works, it is supposed to work. We worked with mixes from three different companies, no differences.

Hi,

Few suggestions to check:

1. Check your copy number calculation and make the dilutions accordingly.(check the Plasmid concentration, OD260nm & 260/280 ratio)

2. Do PCR with Non template control - SYBR, run melt curve and do agarose gel electrophoresis to see possible dimer formation.

3. What is your Amplicon Size? Works well when the amplicon is 100 - 150bp or less.

4. Annealing extension is combined or doing as two separate steps?

5. Probe position from the forward primer can also be checked, closer is better.

6. How well is your amplification curve separated for the serial dilution?

7. Hot Start PCR can yield better results.

-Ajith

Hi Gotham,

if you want to improve your PCR you can also add some (5%) DMSO and try a MgCl2 range between 1 to 5mM.

all the best

fred

Hey Fred the master mix we are using is proprietary formulation of our company and has standardized betaine, DMSO, Glycoerol, and as mentioned above I have already done standardizing MgCl2 concentration by considering 10^6 copy number plasmid as standard control.

I feel the probe quality might be an issue which I have to look into, coz the lower dilution amplification plots were not coming with proper S curve rather in straight line, where as the same primers when used in SyBr green (with out probe) gave better amplification plots in lower dilutions.

Hi Goutham,

Ok, I see that your mix has everything to allow a perfect PCR.

if you want I can test your primers/probe (I use Oligo7), and tell you what.

just send me the sequences of primers/probe and target if you want.

fred

First, I would make a dilution series of the control, make a standard curve and determine efficiency.

If the efficiency is high, then in all probability, the concentration of your control is overestimated.

If it is low, then it is a PCR problem. I don't think it is likely to be your probe, as an efficient PCR will run to plateau level, so the end-point signal strength should roughly the same for all template concentrations.

Have you performed primer titrations? I recommend performing PCR with all combinations of 50, 100, 300 and 900 nM of each primer.

It's often more informative to view your results on a log scale, as this shows up the critical exponential phase of the reaction better.

If you think your probe is giving you trouble, why not check sensitivity and efficiency with the SYBR-green reaction and add in your probe later?

I'm suspicious of GC-clamps. They may be great for sequencing reactions, but in diagnostic PCR they have to be managed carefully, or you get primer-dimers.

Check your primers on Agilent Bioanalyzer to see whether your probe or primers are degraded after the thaw-freezing cycles.

Do controls and SYBR green comparison for control.

Change the Dye and Quencher: I would do FAM-TAMRA (for me it worked)

Change the parameters of cycling (some polymerases, have different activation time).

Target the regions having repetitive sequence per sample.

If you're optimising reagent concentrations then do this on your serial diution panel - not a single plasmid concentration.

I disagree with Armen - TAMRA is the older quencher and the dark non-fluorescent quenchers such as BHQ1 will give better signal to noise. However I agree that your primers should be tested with SYBR. Any sensitivity issues are usually due to primer design, not the probe

I completely agree with all above comments, the issue seems to be primer dimers, I have used premier biosoft free online portal for real time PCR and probe quality measurements, using this didnt give me any significant primer dimers /cross dimers but according to Fred the forward primer seems to have some prominent dimers and hair pins (using Oligo7), So I might have to redesign my primers and see.

Yes Dr. Andrew, I did do primer concentration standardization and probe concentration standardization also the MgCl2 as per the instrument catalogue. I did find some primer dimers in lower dilutions when the real time PCR product and SyBr green product were ran in Agarose Gel. I am re considering desinging new primers, probe seems to have no issue thank you for all the inputs.

Hello Ajith

1. Check your copy number calculation and make the dilutions accordingly.(check the Plasmid concentration, OD260nm & 260/280 ratio)

The purity and concentrations were assessed both spectophometrically, and agarose gel there is no issue wrt to it.

2. Do PCR with Non template control - SYBR, run melt curve and do agarose gel electrophoresis to see possible dimer formation.

I did check with SyBr green and ya the melt curve did gave small primer dimer peaks apart from specific amplification, thus i might have to re design my primers

3. What is your Amplicon Size? Works well when the amplicon is 100 - 150bp or less.

amplicon size is 200 bp.

4. Annealing extension is combined or doing as two separate steps?

For now I am doing 40 cycles of 95-30; 50-30 and 72-30 for probe based, may be 72-30 inclusion is giving primer dimers I shall look into it too.

5. Probe position from the forward primer can also be checked, closer is better.

Probe is 100 bp away from Forward

6. How well is your amplification curve separated for the serial dilution?

Amplification curves are better only till 10^4 dilutions and for later dilutions i do get Ct with a straight line curve

7. Hot Start PCR can yield better results.

I will have to compare it with other commercial hot start PCR mix for it, shall let u know the results once I do .

Thanks for all the queries Ajith

Goutham

Hello again,

Well, John, TAMRA is an old quencher that works well with FAM (life technology handbook for real-time PCR).

Please Goutham clarify your quantification is per what (2000 copies per ml, extract or well?).

Hello Armen, its 2000 copies /ul

ul of extract or in an initial liquid?

Hello Armen, we generally extract cloned plasmid from E. coli and quantify it with Spectophotometer, followed by calculating the copy number of plasmid using Thermoscientific online tool. We would calculate in generally for a stock of 200 crore copy number in 100/500 ul and then make serial dilutions of it till 2/0.2/0.02 copy number (considering lowest dilutions as zero copy number)

From this procedure we generally pick 2X10^5/10^4 (always freshly prepared) for standardizaiton experiments. and for copy number sensitivity assay we perform from 2Lakh copy number to 0.02/ul. (all dilutions will be freshly prepared from mother stock.

Great! So what is your total DNA weight (ng/well) per PCR well in your dilutions

for a 200 crore copy number on an avg the plasmid DNA will range from 1-5ng/ul. The ng of DNA to copy number depends on the plasmid size and the concentration of plasmid DNA isolated.

So what sort of Cq do you get at 200 copies?

If SYBR is working well (and only a few primer dimers detected) then yes, look at the probe. Most of the probe suppliers now use a dark quencher rather than the fluorescent TAMRA in the probe reaction