I would really appreciate any suggestion you can provide.

Hello!! 

I would suggest you to use Reverse Transcriptase PCR so that amplification will be more specific.

Hey Oak,

did you perform a DNA digestion on your RNA samples?

Dear Rangsun Charoensook

First of all, I think if it would be possible the qRT-PCR should be applied.

In my opinion, there are several factors that could cause unexpected results:

1. Reference gene (housekeeping gene): At least two reference genes are needed to have good results.

2. Primer design: the result that you got may be due to the inappropriate bases selection. I think you should try to re-design the primer and check again.

3. The unspecific DNA may be attributable to the contamination or this target gene isn't expressive in this case.

4. Primer-dimer also cause unusual result.

 Hope these suggestions could be useful.

I do agree with Lavanya Deenadayalu that Reverse Transcript should be done prior amplification

First thing I would check is the primer design. Usually it's easier just to make a new set. As mentioned above, contamination is possible. The A260/280 ration can give you an idea. However, I would not suggest doing a q-PCR until you sort out the primer design. Bad primers would output unreliable result, for example, multiple melting curve peaks.

Many thanks for all suggestions, I am trying to solve this problem. I will tell you when I find the reason.

Many thanks again

All of the above answers are to the point. Be extremely thorough when setting up the PCR reaction in order to avoid contamination (include negative control and a positive control namely a previous cDNA that worked or cDNA from Universal RNA-there is a pertinent product from Qiagen). This setup should be followed for both reactions (for the gene of interest which is IGF-1 and for the housekeeping gene). Last but not least clean your pipettes and use filter tips if available.

1. check for DNA contamination.

2. if there is DNA contamination, perform a DNA elimination step (DNase or using DNA filters -I prefer the second-)

3. If there is not DNA contamination, then check for your primer design.

4. If primers show cross-amplification with unspecific sites or produce primer dimers, I would perform 2 new primer designs for the transcript of interest, avoiding unspecific binding and primer dimerization whenever possible.

5. Test the 2 new primer sets and select the one with best results.

6. Finally, you should test several reference genes and use (at least) the 2 best for your assays.

I agree with most of the answers above and see they all are touching on very important points. However, I will try to summarize what they said:

- When you extract your RNA try to make sure that you include a gDNA cleaning steps there are many kits that have this as part of the extraction process.

- Check RNA conc. and quality (there are many methods and instruments). whatever you use make sure that 260/280 is 2.0 which means pure RNA

- When you do Reverse Transcription step (I prefer separately) make sure to include a NO Reverse Transcriptase control where you add everything as your regular reaction without including the enzyme to make sure that RNA is not contaminated with DNA

- When you do your PCR reaction, make sure that you use separate tools and pipette tips to avoid any DNA contamination

- Also include a No Template Control (NTC) to check for DNA contamination during the reaction and presence of primer dimer.

- Don't vortex the tubes as this might fragment you NA and allows different sizes products

- make sure the PCR product length and Primer length are not close so that you can differentiate between them on gel.

I hope I have covered most of the check points.

Good Luck

Many thanks once again for all suggestions.