Hi Shin Wei Tie!

It is not always easy to determine whether the second top is primer dimer or another product. You could try to optimize your reaction by lowering the primer concentration and raise the annealing temperature.

Hi Britta,

Both the forward and reverse primers concentration are 0.5uM and the annealing temperature is 60 deg Celcius. Do you mean to further decrease primers to 0.1uM and increase the Ta also?

Hi Shin,

You could do one thing at a time starting with increasing the temperature by one degree up to 65 deg to see if it helps.

Best Britta

Hi Shin,

May I Know what is the melting temperature of your primers? Before doing RT PCR, did you tried different annealing temperatures for your primers?  Also, have you loaded the RT PCR product on agarose gel (shown image)?

Regards

Sushma

Hi Sushma,

Tm of Forward Primer= 54.4C

Tm of Reverse Primer= 56C

I have just tried of 65C (4th lane from left), it looked like single band but smearing and for 60C (5th lane from left), it looked like having another band around 50bp. I have blast my primers, and it could amplified 6 variants of my target gene. 3 of them are 165bp, 2 are 153bp and another variant, only reverse primer is working. Will it affect the result also?

Dear Shin,

For deciding Ta, I have always tried 4-5 degree less than Tm.

It always worked for me. People also try 5 degree above Tm as there is no thumb rule.

your gel image does not look  very clear. If you do RT PCR on the basis of this Ta, its difficult to analyze the results.

I would suggest you to try gradient PCR with Ta,s lower than Tm ( 50, 52 at least, if possible try 48 and 54.The best thing of gradient PCR is that you can try different Ta in a single PCR run ..:) ) and then run the samples on agarose gel. Once you get single and specific band, then only you should do RT PCR (because its very sensitive, it can detect very minute amount of non specific product)

Hope this works..:)

Best

Sushma

Dear Sushma,

Thanks for the information. I have tried the gradient PCR. Below is the image.

Dear Shin,

This image shows 2 band at almost all Ta, one at 100bp and other one is in between 150-200bp. you said that the product size is around 150-165bp (for all the variants).

So I am assuming that 100bp is non specific (but the intensity of this band is stronger than upper band)??

Am I assuming it right???

Best Regards

Sushma

Dear Sushama,

Yaya, you are right, the lower band signal is stronger than upper band from qRT-PCR also. I have try to check the primer set using Primer Check and the result came back is the forward primers is not working for variant 5. Only reverse primer is amplifying and give around 86bp from the start codon of that particular exon to the last codon of the reverse primer. Thus can I assume this 86bp product also a variant that is amplified also?

But is it okay to proceed qRT-PCR or have to design the primers again?

Dear Sushma,

I checked back my record. There is only one single melt peak and single band on agarose gel for the expression in HepG2. It happened to have 2 bands when I tried out on THP-1 induced macrophage and HUVEC. Does it mean that the variant 5 is not expressed in HepG2 but is expressed in THP-1 induced macrophage and HUVEC (as there was 2nd lower band which might be due to only reverse amplification)?

Hey Shin,

There is a possibility that this band is actually the half amplified product of 5th variant.

But if you are interested in only one variant of your gene of interest, then Its better to design primers again. With these many bands, its not a good idea to do RT PCR ( as you already see two peaks).

Try to design primers which amplify only specific variant. 

If you need some help in primer designing, I can give you some useful  links

Best

Sushma

Also, I can,t say much about the expression of this variant in different systems. Not much Idea.

But from your details, it looks like that its possible. You might would like to have a look in available Literature .

Hi Sushma,

Thanks for your advice very much. May I have you advice on primers' design?

Best Regards,

SHIN WEI

Also, the details on which variants are expressed in my suggested systems are limited. I could hardly find that. If it is so, do I need to determine which variants should I used or I just design another primers set which amplified the same region of all those variants and give the same size of amplicons?

Hey Shin,

For designing primers, you can use primer 3 software. In this software, you can give the details of primer you want ( for e.g. length of primer, GC content, product size etc.). or else can design using NCBI primer design option.  Always design primers on exon - exon junctions.

Do blast the primers to check for the possible amplifications.

After designing primers, you can check the quality of your primer using the below link

http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/.

This links tells you the possibilities of secondary structure your primer can make (Hairpin, self dimer, hetero dimer etc). It also tells you the thermodynamic stability of these structures in term of Gibbs free energy (ΔG).

Till a particular ΔG value, some secondary structures are acceptable. Below link explains this

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Best Wishes

Sushma

If you are interested in total change in the gene expression irrespective of variant type, then its good to design primers which can amplify the common region of all variant.

But if in your experiment only one of the variant is undergoing change, then I guess you would like to study only that variant. If you don,t have idea about this, then you can try first amplifying common region of all variants and see the effect.

Hope this helps!

Sushma

Hi Shin,

It is not allways straight foreward how your melting profile is gonna look like. But you can get an idear by getting a prewiew of your melting profile.

You can allways check the melting profile of your primerdesign using this  link:

http://www.biophys.uni-duesseldorf.de/POLAND/poland.html

Best Britta

http://www.biophys.uni-duesseldorf.de/POLAND/poland.html

Dear Sushma & Britta,

Thank you very much for the advice and suggestion. Appreciate it very much. I will study and try out on new primers. All the best.