I'm doing RT-PCR from rat catilage tissue, and the thing is i searched for tissue specific markers such as SOX9, Aggrecan and type 2 collagen. this is what it looks like, the last 3 lanes are the ones i'm talking about. I was thinking of preparing fresh primer solutions, since the cDNA was positive for actin.
What else can i do?
lanes 2,3 and 4 are b-actin product aprox 350 bp, that's OK. In lane 5 there should be a product of 508 bp, but there's nothing there. I use too much of everything i know, that's why there's so much noise.
Have your primers worked before? Are you sure they are good primers? Are you using gene specific for RTPCR or are you converting your RNA with oligodT/ramdom primer and then amplifying with your specif primers? How much primer are you using (it looks like you have some primer dimers)?
5 minutes of final extension, the primers have worked before. I convert RNA to cDNA using oligo dT. The primer concentration in the master mix is 1,25 uM ( i know it's too high)
i'm gonna start with fresh primer solutions and with a lower concentration for primer and cDNA.
Hi Agustina,
The main issue is that you comment that these primers have already worked properly. Do you mean in an article you read or in your own lab/hands? If it is the first, I recommend to optimise the reaction since the beginning trying mainly a gradient of annealing temperatures and primer concentrations (but if it struggle you too much and you don´t get a quickly optimization, it use to be better and cheaper to design new ones). If you (or in your lab) have already worked, what is the difference between the previous experiment with this primers and the current ones? Check everything…. mRNA extraction protocol, retrotranscription, termocycler…. However, as first trial I agree with you that is best to try new primer solutions and change cDNA/primers concentrations.
Mucha suerte!
How are you extracting your RNA? I've never worked with cartilage before so I don't know how easy it is to process it. Have you checked your RNA purity and integrity? Since Act is an abundant gene it's amplification may not be inhibited while the other genes are.
You should check you RNA 260/280 ratio, RIN and also perform an inhibition curve (use decreasing amounts of RNA to check for the presence of any inhibitor).
i use starndard TRIZOL RNA extraction protocol, and the tissue is squished with the trizol in an homogeneizator (like a tube morter). My RNA had 260/280=1.93 and it showed the 2 band in agarose gel. The primers have worked before here and by my hands haha, but the RNA was extracted from cultured cells, not tissue.
If I'm not wrong cartilage has a lot of polysaccharides and it may act as an inhibitor during PCR. Have you checked your 260/230 ratio? Many polysaccharides absorb at 230 nm, a A260/A230 ratio may be used for their detection: ratios of 1.6 or less are generally considered poor, while ratios above 1.9 indicate very little polysaccharide contamination.
If you have a polysaccharide contamination you should add salt during precipitation to avoid its coprecipitation with RNA. Also, Qiagen has the RNeasy Lipid Tissue Kit that is compatible with cartilage RNA extraction.
https://www.qiagen.com/br/shop/sample-technologies/rna/rna-preparation/rneasy-lipid-tissue-mini-kit/#technicalspecification
https://www.qiagen.com/br/resources/faq?id=10b80501-8bc9-46ba-be68-fc5ec48d5fe8&lang=en
I clean the tissue first and chop it before put it in the homogeneizer,. The tissue is fresh. We sacrifice the rat and take the tissue for the RNA extraction right away.
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