Do you see single melt peak? What master mix do you use for qPCR? Do you first make cDNA? or is it one step qRTPCR? Sometimes it is better to design primers such that products are 200-300bp for qPCR. Also, for 250bp product it is better to run on 1-1.5% gel. 

Is this the first time you are doing a PCR with these primers Shin? Has the PCR been standardized for its reaction conditions? 

It is very likely that PCR conditions are not conducive for a 400 bp odd product to be formed. In addition, you mentioned that you are seeing a smear on an agarose gel. This is strong evidence to tell you that PCR is not optimized and you need to work on it a little more. Please try increasing annealing temperature and you should see the product of the right size. 

For me, yes, it is first time to use these primers but these primers have been used by my labmate. He extracted RNA from HepG2 and I extracted RNA from THP-1 differentiated macrophage. May I know if using same primers but the RNA from different sample does affect also?

I would recomend to make a gradient looking for a temp that gives your desired size using the HepG2 cDNA from your partner as a control of a working reaction. If you can't get the right product in your samples you could try diluting them if you are not already doing dilutions of your cDNAs (sometimes cDNAs drags contaminants that bother with real time PCR). If all this does not work your sample maight be the problem.

Other ideas are:

Have you made a control tube without RT enzyme to rule out possible contaminant Genomic DNA in your RNA samples?.

Does you primers bind in exon junctions? or just in the middle of exons?. You could be having different splicing variants for your amplicon and that could explain the different size.

You can run a primer blast (on ncbi website) to check if you might have unintended targets for your primers. If this is the case you can try using higher anneling temps or adding DMSO to the reactions (o,5 ul for 25ul reactions should be fine). DMSO reduces primer binding, DNA secondary structures and enhances specificity but might lower optimal anneling temperature.

good luck with you PCR!

Make sure that you have no contamination on the reaction mixture (Smear testifies DNA degradation and try to purify the product of PCR reaction). I suggest you to optimize your working conditions, it's possible that the use of polymerases may create mutation (single base pair mutation) which can leads to truncated sequence of DNA 240 bps instead of 427 bps

Guys, thank you very much for your opinion. Herewith I attach the melt peak.

What are your PCR conditions, how many cycles, Ct values,  1 step or 2 step RT PCR? and on what criteria were your primers designed. As stated by Bharat and Ameya having products > 200 bp is not ideal for qPCR. Long PCR products, high copy number, combined with excessive number of cycles can lead to incomplete products being formed.

Dear Ian Teo,

It is a 1 step RT-PCR and the Ct value is 22.  The protocol is as below:

Protocol:

Cycle 1: (1X)

Step 1: 42.0 °C for 10:00.

Cycle 2: (1X)

Step 1: 95.0 °C for 10:00.

Cycle 3: (40X)

Step 1: 95.0 °C for 00:10.

Step 2: 60.0 °C for 00:30.

Step 3: 72.0 °C for 00:30.

Data collection and real-time analysis enabled.

Cycle 4: (1X)

Step 1: 95.0 °C for 01:00.

Cycle 5: (1X)

Step 1: 55.0 °C for 01:00.

Cycle 6: (81X)

Step 1: 55.0 °C-95.0 °C for 00:10.

Increase set point temperature after cycle 2 by 0.5 °C

Melt curve data collection and analysis enabled.

There does not seem anything obviously incorrect about your conditions. I generally do a 2 step PCR with random primers and a longer RT time and use the diluted cDNA.  10 min may be a bit short for RT depending on the processivity and complexity of your RNA (most RTs have a processivity of 20-40 nucleotides, some  eg Thermo-Fisher Maxima claim to be able to synthesise up to 7.5 kb in 5 min). Other protocols suggest 30 min. A simple expt would be to increase your RT time.

A Ct value 22 does not suggest that the system is overloaded. However, I assume you are collecting data at 72C. It makes much more sense to collect it nearer the peak Tm (Tm = 85, collect data at 80-82). If you look at the area under the peak, a lot of your signal is generated by the hump on the left side of the melting curve between 72 and 82C. This is not amplified target ( could be DNA?) and will give you a wrong Ct value.

A smaller amplified product size will be helpful.

You may want to check the integrity of your RNA and incorporate a DNase digestion step if you do not already in your RNA prep. Also try someone else's RNA as a check.

Dear Ian Teo,

Thank you very much for the reply. May I know is it to increase the RT time in one step qRT-PCR from 10 min to 20 min perhaps?

What do you mean by data collection near to Tm peak? Is it for the melt curve analysis, I just enable data collection from 82 to 95C?

I would try to increase your RT step to 20-30 min.

I assumed that you were acquiring fluorescence at the 72C step for each cycle in step 3 in order to generate your amplification profile and Ct values (though you do not state your acquisition temperature). You need to incorporate an additional step to measure fluorescence nearer the Tm of your target.  eg 

Cycle 3: (40X)

Step 1: 95.0 °C for 00:10.

Step 2: 60.0 °C for 00:30.

Step 3: 72.0 °C for 00:30.

Step 4  82 C for 10sec with fluorescence acquisition.

By measuring Fluorescence at 82C you will eliminate all the unwanted non-specific signal generated between 72C and 82C  which you see on your melt curve. This will lower your total fluorescence measurements and therefore change your Ct threshold. At the moment you are measuring both specific and non-target  fluorescence.

There is still the issue that you are not getting the correct size of amplification products - 427 bp which will need to be addressed by modifying your RT and amplification conditions.

My advice would still be, that if you can, to go for another set of inter-exon primers to give you a product between 120-200 bp. Note that above 200 bp the melting curve profile of your product will usually be >85-90C  and you cannot easily discriminate between a product of 200 bp vs one of 400 bp,  whereas between 100 and 200 bp you can make a distinction based upon product Tm. 

To give yourself an idea of the resolution of your melt analysis, you can always take PCR amplification products of say 100, 150 and 200 bp (as determined on gels), add them to Sybr Green mix and do a melt curve analysis. You should see a peak Tm shift to the right with increasing length of product.