Dear Arefian,

before i suggest anything to you i would like to know more about the expected amplicon size and the size of ladder used in the gel. how much DNA you took as template and primer concentration and also how much you loaded in gel.

After extraction of RNA, you must give DNase treatment to reduce the amplification from genomic DNA. And check the primer with Primer blast in NCBI if it binds with more than two genes or may be the gene you are trying to amplify has many isoforms, and primer bind with other genes also.

good luck

Frankly, there is no easy answer.

For how many cycles did you run your PCR?

How much cDNA was your input?

The simplest explanation could be that your gene is not expressed by your cells and performing many cycles you just obtain junk amplicons of unrelated transcripts with partial matchings to the primer sequences.

Try another set of primers and check the primers for complementarity (

To reduce unspecific products you can also vary the concentration of MgCl2 in your PCR solution, which usually is 15 mM in the 10x buffer. The higher MgCl2, the more unspecific the amplification. If you have a PCR machine with gradient function you can test several combinations of annealing temp + MgCl2 an once.Most PCR kits also contain a buffer without MgCl2 where you can add the concentration you need.

What strikes me, though, is that your "unspecific" products are very strong, especially in lane 3. Usually, unspecific products tend to be fainter. Therefore it really might be a problem of different splice products you have here. What product size do you expect anyway from the cDNA and gDNA? Is the band in lane 2 the right size or might in be just primer dimers (often visible around 80 bp) because your primers can't amplify from genomic DNA (often the case when the intron in between is too large). You should check in a database for the sizes of the introns in your gene (e.g. www.ensembl.org).

Greetings,

Britta

Hi,

Addition of DMSO (5-10%) in the PCR reaction would increase the specificity of the primer annealing.

Did you blast your primer sequences? If your primers are not specific, re-design them, perhaps you can start with two different sets.

other things you can consider are changing the primer concentration, annealing time(5-10 secs), decreasing the extension time (not the final extension). 

Hope that helps!

Dear Arefan, 

You probably should check these aspects:

1. Verify your primers - if is correct, if annealing between themself, amplicon size and if is specific;

2. Check your samples - did you make a good extraction? (put a negative control too);

3. Dilute your sample in case they are ok;

4. Try an PCR gradient with diff temperatures.

Hope helps.

In addition, (if the above suggestions are not sufficient) and If the sequence of your amplicon is known, you could try non-cutting restriction enzymes to digest unrelated sequences. To be sure you don't eliminate amplicons originating in alternative splicing, I'd have these bands sequenced however.

In addition to the above ,I think you can try by  decreasing the cDNA concentration or say diluting the cDNA as well as you can try out by decreasing the no. of cycles for RT-PCR.One step RT-PCR is also which you can try out.

use Tetramethylammonium chloride (TMAC) if you can get your hands on it!  a concentration of 15-60 mM, this can also increase the annealing temp of at rich primers, which increase the pcr effiency 

Try to reduce the primer concentration, be stringent while designing PCR primers.

Also you can design the primer which spans the two exon regions so that the chances of amplifying the intron region (occurring from Genomic DNA contamination) is avoided.......

At least you should find one band in lane 3 and 4 to be the same size as lane 2 with genomic DNA. Since this is not the case I think the bands in lane 3 and 4 are aspecific, maybe some internal priming has occurred with degraded small RNA or DNA fragments, maybe  ALU sequences that are highly aboundant in the genome.

You can analyze the quality of RNA using Agilent Bioanalyzer.

Try to reduce the extension time which may eliminate the larger fragments. Always include negative control to ensure that no contamination has occurred with PCR reagents. You may first use the constitutive control genes like actin to standardize the RT-PCR. Double check the primer to ensure specific binding.

Why can you useeither random hexamrs or oligo dT for making cDNA ? In quantitation used lable probe with in your gene specific primers. I think that will help. 

Try to reduce concentration of Mg2+ in PCR mix. Lower concentraion of this ion leads to higher specificity, but lower outcome and vice versa. I also would recommend you to check your primers by Primer-BLAST tool to see what else can be amplified. 

you are getting your desired band in Lane 3 and Lane 4, the problem is only multiple bands. This could be due to over amplification of gene, High concentration of Primers or Dimerization of amplified products due ploy DT and PolyA. So, Please review you undesired band size,if it is double of your desired band this may be the case.

to rectify this, please be stringent with amplification time, use optimum concentration of primers and play with concentration of Mg also.

And before loading on gel, you could pre-warm the product at 70 C.

1. Change the primer and design with NCBI or primer3 software.

2. try to include intron in the amplicon means the primers should be in two different exons spanning an introl of at least 1000nt.

3. reduce the amplification time like if your product is ~200bp, 10-15 sec of amplification will be enough.

4. reduce the primer amount to something like 1pmol for 10ul reaction.

any of these should work.

Good luck