Another phenomenon I observed was after I have stained the gel with ethidium bromide, the bands looked faint after few minute. Is the ethidium bromide having problem? But the ethidium bromide is fresh and is orange in colour.

Hi Markus.

I did not have positive control.

Below are the primer sequences:

Forward primers: 5'-tca ttt tga cgg aaa tgg caa cc-3'

Reverse primers: 5'-aag gaa cat agc ttc aac cgc ag-3'

Dear Shin...so it seems that you have specificity problems...first, how clean is the substrate and how much did you used? how do you extract your RNA and how you retrotrascribed? OligoAnalyzer http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ show a Tm of 56°C for one of your primer...check this with your conditions (Mg or Na conc). Also you can try to add Mg to your mix..

so..for troubleshooting:1- increase the Tm up to 67° and see if you get some sharper bands. 2-if you do not get any better, fix one the temperature (i.e. 64.2 seems to give good results) and change Mg conc

For EtBr: did you left the gel on plain white light or (worse) under UV? remember that the signal get faint after some exposure....

Dear Fulvio,

Thanks for the advise. I am using 100ng RNA per reaction. The substrate is clean and I did one-step real time RT-PCR, which mean RT in the same tube then only proceeded to PCR. I cannot adjust Mg concentration as is premixed in the mastermix. I am using Quantifast one step kit from Qiagen. 

If fixed to 64.2, i have 2 peaks. Is it okay to proceed with gene expression? 

Dear Shin

so..what you mean by substrate is clean? 260/280 >1.8? is that correct? 

you can try to increase the starting RNA, going up to 500ng in step to check if your specific band increase...could be that your mRNA is low expressed. Try also to find a positive control (i.e. cell line/treatment taht would increase your G.o.I.) that will be specially important for gene expression measurements

also I would try to increase at least to 65°C to check if you get better specificity..I would not recommend to make assumptions about gene expression with 2 peaks (as the one I saw..)

Yaya.. My 260/280 was 1.91. I have done positive control in HepG2  cells. Yes, the current model of THP-1 differentiated macrophage has low expression if compared to HepG2. 

Yup. You are right. I also do not accept with 2 peaks for the gene expression. But I have tried more than half year to try on this. Previously I tried on another primers which amplified DNA binding domain of the nuclear receptors but one of the primers is not working on one of the variants, so giving another incomplete amplicons around 90bp (167bp for other variants). Thus now I design another primers which amplify ligand binding domain. Sad to say the result showed something not as expected and I already lost idea to figure out. Perhaps to really get helping hand from all of the experts here. 

Okay, I will try another run for increasing Ta. Thank you very much. Have a nice day.

These were the run that I repeated this morning.. I am yet to run the gel.

Hi Shin

First at all, you should check not only the purity of your RNA, but its integrity, for example, in agarose gel. 

In your case, it seems there is no specific product. The bands could be primer dimers or non specific amplified products. In this case, I should design new set of primers, if it´s possible several pairs (cheaper than wasting your PCR kit in doing many tests). Some of them should work properly and give you the right band. To design the primers, try to use a program. Even with a program, you can get a bad pair of primers, that happened to me several times. So that´s why I like to order several pairs for the same cDNA fragment to be amplified. Choose the parameters that are needed in real time PCR (Tm, GC content, etc). To test the primers, use first a cDNA control that you know  should give a specific product after PCR. This control could be a plasmid containing your gene of interest or RNA isolated from cells that actualy show expression of that gene.

Make sure also to include controls in your reaction: non-template control and no RT control.

Good luck.

Your melt curves have multiple peaks and your gel picture shows multiple bands in a sinle lane. Multiple peaks in RT PCR graphs arise due to multiple band amplifications in a single reaction. Please standardize your primers first and then run RT PCR.

i've had some problems with this happening to me.

here's some of my experience:

1 - good controls, which means where are your housekeeping genes to start with? Your RNA integrity should be checked there, not only on a nanodrop, which can give false reads.

2- Dillute your RNA to the final concentration not your pre-set target concentration. For example, if you are adding 10 ul of the final mix to 5 ul of RNA, you should dillute your RNA to 150ng/ml, not 100nl/ml, since in the end you will have 100ng/ml.

3 - try to optimize your cycle. Sometimes cancer cells are so messed up with over expression of some genes and under expression of so many others, that your gene may amplify before or later. even with two peaks, you can tell you are in the right way, because there are only two peaks. a major problem arises if you have multiple peaks. you still may have iy, but you have to adjust your rt-pcr cycle so to amplify one major peak only. than means if it amplifies before the melt curve you wish, you should change temp or diminish a cycle, whereas if it amplifies later you should raise temperature and optimize cycles.

4 - if you have tried everything, like myself (I spent almost 2 years trying to make this right) there is a light: from the start, make a curve with rising concentrations of your stimuli (i.e. LPS or anything else) and also one with time per concentration. Start with 2 hours, then make 4, 6, 12 and 24 hours and see if there is a difference in the amplification pattern. That should do it (it did the trick for me, btw).

I have done primer optimisation. 

Lane 1: 50bp Ladder

Lane2: 300nM Forward, 150nM Reverse

Lane 3: 150nM Forward, 300nM Reverse

Lane 4: 150nM Forward, 150nM Reverse