Looks like genomic DNA contamnination. gDNA runs at about the same position regardless of the gel %, i.e. high in the gel so the size is independent of the markers. Did you DN'ase treat the RNA before RT? How much starting template did you have in the PCR reaction? As Aparajita said, its pretty faint anyway, so ignore?

Hope this helps.

Good Day Marte,

I am not able to download the picture of your gel, however from my experience with gels it is possible that you are seeing a primer dimer (which is typically 30-50 bp). If that is the case it can easily be corrected by optimizing the amount of primer added to the PCR reaction. It is possible that the primers are binding to each other and this is the band you are visualizing on the gel.

What I find slightly strange though, is that if this is in fact a primer dimer you should have seen a peak on your melt curve analysis, the peak would typically have a lower melt temperature than your product.

I hope that this helps you.

All the best!

Dear Marte,

I took a look at your gel. I do not see any primer dimer bands in there. However, if there was a single peak in your melting curves, it is highly unlikely that you have other amplicons in abundance. How much cDNA did you use for the qPCR and what is the cDNA length?

I don't think it's a primer dimer because it is not at the bottom of the gel. I used a 0.1-10kb ladder and this unexpected band is above my 10kb mark. 

As far as how much cDNA I added: I added about 150ng of RNA in a 40ul reaction. Then, I added 2ul of my cDNA to my qPCR reaction.

Dear Marte,

Your quantities do not seem over the roof. also it is highly unlikely that qPCR amplicons are that large. I have a feeling that these are non-specific DNA...possibly contamination, and for all you know it may not be DNA, since it did not turn up on the melting curve. Most qPCR reporters bind to dsDNA/RNA or helical DNA/RNA structures, so ssDNA/linear may not be detected. Now if you are using a cocktail of SYBR dyes for band detection in gel, then those are capable of binding RNA or non-helical structures as well. And for how faint it is, I would not worry about it. Moreover there are no bands in the area of MW size you are interested in. 

Hope this helps

Regards

Looks like genomic DNA contamnination. gDNA runs at about the same position regardless of the gel %, i.e. high in the gel so the size is independent of the markers. Did you DN'ase treat the RNA before RT? How much starting template did you have in the PCR reaction? As Aparajita said, its pretty faint anyway, so ignore?

Hope this helps.

Hi, I don't know if you verify the quality of the RNA before doing your cDNA. What you have at the top of the gel looks like genomic DNA contamination or non denatured RNA. As for your positive control you have only the expected band your data should be fine.

Hi

I agree with David A Mann, looks like a slight genomic DNA contamination. I would not worry that much about it.

However, the bands of your positive sample look very different to me (at least lane 1 compared to lanes 5 & 9). I am assuming they are the triplicate so I would check the melting peaks again to see if they completely overlap and, maybe, even sequence those 3 PCR products to be sure you are amplifying the right product.

Good luck!

Alfonso

I also agree it's gDNA contamination. It appear to be high molecular weight and a small amount. And I agree it probably is not interfering with your qPCR as long as you have well-designed primers that won't amplify genomic DNA.

Hi

I also agree it looks gDNA. I would try to get rid of it if you want to have a more accurate experiment .

The Genomic Dna is probably in all the RNA samples that you have. 

If you want to be completely sure, run a standar PCR with your RNA samples and you can check it.  If you have any amplification means that you still have DNA in yor RNA amples. 

As David A Mann says, did you DN'ase treat the RNA before RT? 

All the best!!

Hi Marte, 

As it was said before, it looks like that the high band may be genomic DNA that is not able to pass through the gel because of the size.

Usually after RNA extraction you measure the quality and amount of RNA (Nanodrop) the ratios should be among 2.0 (260/280) because of the uracile present in the RNA. Lower or higher than 2.0 values may represent some source of contamination. Either way is better to treat the samples after RNA extraction with genomic DNAse kit (quite cheap and effective).

I hope it helps!! :) 

Carlos. 

You can treated RNA with DNase I.

Dear Marte,

What i understand from your description is that you have treated your RNA with a few molecular techniques before running the attached gel (RNA -- cDNA -- amplified DNA). By experience, each step is not perfect and so you have some carry over of unwanted products. In your case it might be a really long RNA strand which was present right from the beginning and thus was reverse transcribed to DNA (its only a hypothesis). On that note, did you use a PCR purification kit? That might actually get rid of your heavy bands (noise). But then again since those heavy bands are from your negative samples, they weren't amplified so if I were you, I wouldn't really worry about it. 

Hope this helps.

I also agree that with such multiple steps no single step is perfect. However, there should be an ultimate goal for your construction. You may be better of focusing on extracting the product that looks like what you want, purifying that and using that for further steps like transfection. Otherwise, you will need to optimize each step before going to the next one. Optimize RNA extraction to avoid DNA contamination. Optimize reverse transcription either oligo dT or random priming. Optimize PCR amplification, usually primer construct to avoid dimerization or non specific priming. All the best.

Hi Marte

I think your first step would be Re-do the PCR purification steps.  Observe the results.  Then use a optimizer for your PCR amplification if the purification steps did not improve the samples enough.