Dear Zahidah, 

As you are using Taqman probe, you cannot check the denaturation profile. So, I would recommend you to run electrophoresis in order to be sure you have a single band as a pcr product.

Second, please check that the crossing points are in the acceptable range, so you dont have an excess template.

Third, it is still possible that your template has inhibitory compounds, so I would run a standard courve with a cloned target gene, in plasmid form.

Try to rule out some of these possibilities, and contact me if you still have trouble. 

Yours, 

Purificación Carrasco

And be sure that the primers concentration are around 300 micromolar.

Sorry, primers final concentration around 300 nM

Without out knowing your exact details, it may be that your very 1st point is template- inhibited (too much sample/too much target template).  And this problem would be even worse with 18S since it is always about 250,000X more abundant than most other transcripts - so your curve for 18S is probably showing template inhibition in the first 2 or 3 points in your curve.

The pre-made Taqman assays you are working with use 250 nM probe and 900 nM of each primer (which is usually overkill). Whereas 125 nM probe and 450 nM of each primer (in the final reactions) works just fine in most hydrolysis probe-based qPCR situations.  Save your self money and cut your primer-probe mixture addition in half, and start your curves with less template as Purificacion has alluded to.  But, realize that the 18S curve will still have to start at 1:1000 more dilute than most other targets...  Good Luck.

If you made the dilution of the template solution how you describe (1 ul template and 19 ul sterile ddH2O), you are wrong. It is not a 1:10 dilution. It is 1:20 dilution. Try to fix it

Hi Zahidah, if you are using a one-step mastermix (RNA as template into RT-qPCR mastermix) you have to take into account the RT efficiency as well as PCR efficiency. If you wish to look at PCR efficiency only I would suggest carrying out a two-step RT in which your RNA is converted to cDNA*, then prepare a standard curve using dilutions of this cDNA to examine if it's the conditions of the PCR that are suboptimal. If this works out then you need to re-examine your RT conditions. 

Like PC mentioned, a cloned target in plasmid form would be the best material for DNA standard curves or a purified PCR product (ideally prepared using a proof-reading enzyme).

As you mentioned that you used a control gene (18s) but got the same results as your target it may not be related to problems with your template, but as a precaution, if possible, extract your nucleic acid at high concentrations and dilute as necessary so that inhibitors will be sifted out but your template will be in high copy numbers.

Any additional details regarding your experiment (RNA/DNA, source of material etc., nucleic acid extraction method) would add value to your question.

* you can use a dedicated two-step RT-kit with the same RT enzyme as your original one-step MM or carry out RT using the one-step MM and deactivate at 70-80C range without activating the DNA polymerase - then dilute the cDNA for a 5-point range

Is the template DNA or RNA, which organism is the source of the material, and what is the target to be quantified?

How was the template prepared?

How many technical replicates are performed?

What are the R2 values and slope of the STD curve?

If your R2 value is poor, that usually indicates a problem with your pipetting method, or you have reached the dilution limit of your template if it's only happening in the most dilute couple of samples.  If a couple different probes are showing poor efficiency, this usually indicates your template is contaminated with organic compounds from the extraction process; did you check the A260/A230 of your template?  As others have mentioned, the interpretation is complicated because you used 18s, which has it's own issues due it's extremely high concentration.  Seeing a graph of your standard curve could help.

I immediately had the same response as Ilya.

If you make your dilution curve as described, you are making a 1/20 dilution, but labeling it a 1/10 dilution. Re-enter the correct values of the dilution in the software so it knows it is a 1/20 dilution or remake your std curve for a real 1/10 dilution.

With your current settings it is normal that your R² is not good

sorry my mistake. For template,I have done 1:10 dilution, (2 ul template and 18 ul sterile ddH2O) and I dont know why I cannot attach my standard curve graph here.very sad.

For my target (squalene synthase gene from 10 years old root tongkat ali),

-slope : 0.144

-y-intercept : 34.84

-R2 : 0.051

Those are some concerning numbers.  Do you mix each well of the reaction separately or make a master mix?

I will add one more point to the preceding fine suggestions.  To make serial dilutions, increase the volume to limit stochastic effects of pipetting ie., 10ul template and 90ul water for multiple dilutions.  Also, use "large" volumes when setting up PCR eg. 10ul template (appropriately diluted of course) and 40ul of Master Mix.