We have cell lines that were obtained from patient tumors. We would like to validate their origin using a cell line authentication service or similar. Does anyone have experience with this?
genotype both the cell line and your patient samples.. I think like genetic fingerprint sequencing. the way police searches for the criminal identity. there PCR kits which can do that.
DNA fingerprint! There are labs that make this kind of services if you don't have equipment or experties.
I agree with the two previous answers. We have recently published a paper for quality control ina biobank using a simple and low cost method to double check the
Sorry I closed by accident my answer.
If you go to pubmed you will find a paper on:
Int J Biol Markers. 2013 Jul 19:0. doi: 10.5301/JBM.5000044. [Epub ahead of print]
A reliable and reproducible technique for DNA fingerprinting in biorepositories: a pilot study from BioBIM.
Palmirotta R, De Marchis ML, Ludovici G, Ialongo C, Leone B, Lopez N, Valente MG, Spila A, Ferroni P, Della-Morte D, Guadagni F.
Using a DNA fingerprinting protocol you provide assurances of DNA identity and quality in a biorepository setting. THis cpould also help you.
They are right, you should genotyping the cell lines' DNA against the patients'. We are running a biobank and I can share some experience with you. The following is the PCR kit you can use "the AmpFℓSTR Identifier PCR Amplification kit (Life Technologies)". It is a standard kit for cell line authentication.
The principle is PCR-based microsatellite typing. There are repeated DNA sequences in our genome, the numbers of some of these repeats vary from person to person. Eg. You can carry 5 repeats for a particular locus and I have only 3. We use PCR to amplify the surrounding region of those repeats. So the PCR product of your genome is 2 repeats larger than mine after running them on a DNA gel.
In reality, a genetic analyzer is used instead of running all the samples on a minigel. The readout for the PCR products is peaks but not bands. To match the DNA of a particular cell line with a patient, the patterns of various repeat PCR products (microsatellite markers) of the cell line’s versus the patent’s will be compared, and to see whether the two patterns are identical.
What the PCR kits contains is mainly the PCR primer pairs, the conditions are optimized by the manufacturer. What you need to do is PCR the DNA with the primers from the kits and send your PCR products to a DNA analyzing company such as BGI. Of course, if cost is not an issue for you, ATCC or other biotech companies will provide genotyping service, all they need is the purified DNA and they will perform everything for you. I hope my humble advice can help you. Good luck!
You can refer to some of our papers for reference:
1.“Cheung et al. Polo-like Kinase Inhibitor Ro5203280 Has Potent Antitumor Activity in Nasopharyngeal Carcinoma. Mol Cancer Ther.12(8):1393-401” ,
2. Lung HL et al. (2004) Fine mapping of the 11q22-23 tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma. Int J Cancer 112:628-35.
thank you all for these answers! Is DNA from FFPE material good enough for these assays?
Depends on how long the FFPE tissues have been stored and how the tissues were fixed and prepared during the FFPE treatment. We tested FFPE samples from different hospitals, FFPE samples from some particular hospitals can consistently results in poor DNA quality. This is nothing much we can do because the samples were already prepared .DNA fragmentation is an issue for the FFPE materials. PCR products less than 200 bp should be fine for amplification, I think the authentication PCR should work fine. All the best!
You can send some DNA to facilities such as MD Anderson in Texas they're really good. They can check it against their database of cell lines and give you a match. I agree that DNA from FFPE gets fragmented, but high throughput sequencing works only for fragments anyway, so it should not be an issue.
I also have an excellent protocol for FFPE extraction. Just ask me if you need it and I'll send you a copy.
We just began using the Sequenom MassARRAY system for DNA fingerprinting at our BioBank. It is suitable for FFPE tissues and would quite easily validate your cell lines. If you can find someone close with one, they should be able to help you out as it is reasonably inexpensive.
Hi Paul,
if you are looking for a good service, I would recommend the DSMZ (German collection in Braunschweig) - should be easier to send the samples since you are European-based. Price for scientific people is about 150€ per sample.
In my lab, we perform a finger-print using an in-house PCR protocol + fragment analysis adapted from commercial kits like the one Hong suggested - if you are interested, I can send it to you. Works fine for research - and there were no discrepancies when compared to a few samples analyzed from DSMZ.
Hi Paul - yes, I would say that STR DNA profiling would be the most straightforward, simple and most cost-efficient method for authenticating your cell lines compared to the patient of origin. The added bonus of STR profiling is that it is a data output that is the same no matter what lab runs the sample. I work for a company in the USA that accepts samples from all over the world - our service runs as low as $70/sample (discounts available for volume) - but we also offer many different report options which allow you to use the data as you need it (whether for internal quality control or for publication). Our website is www.celllineauthentication.com - or feel free to contact me for more information - I would be more than happy to help you get the testing that you need for your samples. Good luck!
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