You can check your cdna by running it on 1% agrose gel which will give you a smear, this will assure that your cdna has been formed. Alternatively you can do a qpcr for known gene.

I suggest running REAL TIME PCR on a gene that you know should be regulated with your treatment. Also, run the appropriate house keeping gene.

Its sounds like you want to try semi-quantitative PCR. There is no way of knowing necessarily which gene is best. The best way is to look up in literature to see which housekeeping gene people use for your treatment(this should be a good indicator of which housekeeping gene, but you should test multiple housekeeping genes). Here is a website that has a good introduction and protocol for the technique. http://stanxterm.aecom.yu.edu/wiki/index.php?page=Semi_-_quantitative_RTPCR

I do suggest a more quantitative method such as quantitative Real Time PCR, which involves more sophisticated machinery(rather than running a gel).

Thanks. But what about using the primers of housekeeping genes like Actin & 26S. Which one among the two is more reliable and if they don't work, can we still call cDNA to be ok. Besides, how much cDNA has to be loaded on the gel. Adieu

Well, the RNA that is still present in your cDNA (unless you do a RNAse digest) will also give a smear, how would you discriminate it from your cDNA?

Photometry will not work either since the RT mix will obscure your measurements, which means you would have to clean your cDNA beforehand. Again, you would get wrong absorption measurements unless you remove the RNA.

In my opinion the only reasonable way to check cDNA is to measure some reference genes.

There is an assay using the ratio of the 5' and 3' ends of the HPRT gene to assess initial RNA integrity. If you measure that ratio, it should remain constant over time, unless your cDNA starts to fragment. Or you could design an assay using a long (>1 kB) PCR product. The more your cDNA fragments due to freeze/thaw/aging/etc. the lower that signal should become.

But I am not shure why you want to check at all. If you always use the same amount of RNA, you should always get a similar Cq (quantification cycle, sometimes named "crossing point") for a given tissue/cell line with constant proportions of cell types. If you measure your reference gene(s) they should be within +/- 1 cycles and everything should be ok.

From my experience cDNA is quite stable at -20°C (> 5 years, ~5 freeze/thaw cycles), if you aliquot it undiluted after RT you should not have a problem with your samples even after some years.

see http://www.kpl.com/docs/datasheet/600600.pdf

It is an assay measuring the 5' and 3' end of several ubiquituous genes up to 6 kB

If you want to use 26S you have to RT your RNA with random hexamer primers since this RNA has no poly-A-tail. However, the use of 18S/28S as reference gene is strongly discouraged since it is a) no mRNA and b) far stronger expressed in cells than any mRNA, i.e. the 28S expression allows no conclusion on any mRNA. Use b-Actin or some other genes like RPL27/37/41. If Actin is appropriate depends on your cellular system. For me b-actin is useless since it is regulated by TSH and I am a thyroidologist...

There are metaanalyses available for various kinds of tissue/cell lines, most I have seen recommend a gene from the RPL family, sometimes UBC or CALM2. But it depends on the cells and the treatment.

I think that the firs step to evaluate newly synthesided cDNA is to be certain that you really have such cDNA. Shall an agarose gel electrophoresis may be helpfull?

I use first of all a photometer, but you must not stop there. Even a blank sample will gives you results (260/280=1.7-1.9). So I check everything with agarose gel. Alternatively you can use specific housekeeping genes in qPCR. I suggest to test also the 5'-3' parts of genes. I have noticed in many experiments, that for small products (150bp) i got good results in PCR, but i had no product for longer. So you can design primers from 5' to 3' of your housekeeping genes.

Once upon a time when we were making cDNA phage libraries from polyA preparations s we used to make a small amount of radioactive reaction and then just load an agarose gel with a reference ladder (also labelled) to see what was the average size of the transcribed cDNAs. Fast and easy: best cDNA preparations should have averaged around 2kb with little smear below this size.

Use agilent bioanalyser if you want cDNA for sequencing etc

Guys,

I made a cDNA with High capacity kit and ran an agarose gel (1%) to check, BUT...there was nothing!!! I didn't had a single band, and not a smear either! Anyone have any idea what may be happening?? Because after that I ran a qPCR with housekeeping gene and another gene of interest and I had amplification! The results were ok, with good amplification and a single melting curve.

Any ideas?

Concerning cDNA integrity checking. We described a single multi-purpose PCR-based integrity assay amplifying multiple fragments: doi: https://doi.org/10.1101/168195. Don't hesitate to contact me for feedback.