For your info Nallasivam, I'm study TERT gene which located at 5p region of chromosome 5 in leukemic cancer cell line....in early passage, amplification of TERT gene varied with majority showing 3 - 6 copies in TERT gene. After 20 passage (late passage), majority of the cell showing trisomy of chromosome 5...

As you told there is Trisomy after 20 passages then TERT copy number should increase. However, you are not getting any amplification. I think you should check whether your experiment is working fine or not . Can you please write some detail about your real-time experiment ?. I assume that you are also putting some internal and external control along with your targeted gene and if your controls are working fine but you are not getting any amplification with TERT gene then you can try another primer sets specific to gene linked with your TERT gene at "P" arm of Chromosome no. 5 and then you can conclude in a better way.

Thank you Nallasivam Palanisamy....for your info I'm maintaining cell using RPMI 1640 medium, FBS and penstrep antibiotic without exposure to colcemide solution. As far as I'm concern, the function of colcemide solution is to arrest the mitosis at metaphase stage. Since, I'm doing interphase-FISH analysis,the use of colcemide solution is not crucial. Do you agree??

Hi Sandeep Rajput, I'm using FISH and qPCR to determine the TERT gene copy number status. For both of the analysis, the result concordant... In early passage cells, the ratio target gene copy number to reference gene copy number was more than > 1. It mean the target gene copy number increased. In late passage cells, ratio target gene copy number to reference gene copy number was equal to 1. It means the copy number of targeted gene and reference gene was the same.

Gene expression changes are common in young and late passage cells. It is recommended that experimental procedures should be conducted within 10 passages post recovery. We had similar problems with HMEC cells that showed completely different levels of gene expression at pass 10 than at pass 20. You will need to conduct your experimentation within a small range of cell passes to gain reproducible results

Thank you Lynn. I will conduct my experiment according to your recommendation.

@Lynn Mccallum

As far as I understood the question of Syibli Othman the problem is/was copy number alterations not gene expression.

And I have a probably very nasty question: Is there any chance that your cell line is contaminated with another cell line, which is growing faster and in later passages overgrows the cell you actually want to analyze? Please, don't see this as offence, I am just asking, because I know too many cases were cell line contamination was the culprit for irreproducible data.

Can I ask, if you are performing interphase FISH analysis, how are you differentiating between intrachromosomal amplification of TERT and trisomy 5? Is there an additional FISH probe? I actually think it would be worth performing metaphase FISH is you haven't already. It will give you much more detail. Maybe you could also perform array-CGH at the two time points, this might help resolving the disconcordancy.

Also you mention a normal Ct value in late passage cells but the presence of trisomy 5. This seems contradictory. Maybe a silly question, but is you reference qPCR gene on chromosome 5, or another chromosome targeted by aneuploidy or rearrangement?

Last point. You are doing quantitative genomic PCR, right? It is often quite difficult to detect subtle changes in copy number using this approach. How many TERT specific assays are you using, and how many technical and biological replicates are you performing? It is often worth having several different assays targeting your region of interest, and performing 5-7 replicates to accurately detect a shift in copy number.

Many studies have uses three distinct qPCR assays across a contiguous region within you gene of interest.

I hope this helps.

Sabine Strehl,

It is not offensive to me. As far as I concern, based on the morphology and characteristic of the cells, I pretty sure I'm maintaining the right cell. Besides that, the lab that I'm working right now only maintaining one cell line and I assume chances to have contamination with another cell line is low. Yes, I would agree with you with the cross contamination problem in cell culture work and I have put highly prevention effort to it.

Jon Strefford,

I only performing interphase FISH using dual color FISH probe (5p51 for TERT / 5q31 as reference) which both probe located at the chromosome 5. By using dual color probe, we can differentiate between intrachromosomal or extrachromosomal amplification, right? I do not proceed with metaphase FISH since the FISH result was concordance with qPCR analysis result. However, I will perform metaphase FISH after this to reconfirm the result.

In qPCR analysis, I would like to obtain the gene copy number ratio by dividing the quantities of targeted gene (TERT) with quantities of reference gene. I'm using gene located at chromosome 15 as a reference gene, since gains or losses on this gene are rarely or not observed in the cancer cases that I studying right now.

Yes, I'm doing quantitative genomic PCR. I'm using Touch down PCR method to prepare PCR product which will be used to construct the standard curve in qPCR analysis. Before that, the PCR product will be detected by using agarose gel electrophoresis to confirm the product size and quality. Is it consider as a specific assays? The qPCR analysis was done in triplicates for each samples as recommended by the previous studies.

Regards.

All sounds good. You are quite correct, that you FISH probes should differentiate between aneuploidy of chromosome 5 and non-aneuploidy based TERT copy number gain.

I think it's critical that you perform metaphase FISH, to sort out your issues, probably using a chromosome 5 painting probe and your TERT specific probe (if you have it as a single colour FISH probe). If not, then you dual-colour probe on metaphases. I would do this at both time points. I would also do some chromosome 15 painting (or a probe specific for your reference gene, if possible) just to answer the question of you qPCR control probe. Using a reference gene based on primary tumours when you are using a cell line might not be the best approach, as cell lines exhibit so much more genomic instability. It might not be an issue, but worth checking.