I was hoping that some of you might be able to help me set up an experiment in which I want to measure telomere length of MNCs (of a certain patient population) by Flow-FISH. As I'm completely new to this I'm running into a whole bunch of issues. If you have thoughts on any of them, feel free to comment.
First off, what probes are best to use? Traditionally people use PNA probes, but Exiqon also seems to offer LNA probes these days, are those any good? Others say Bridged Nucleic Acids (BNAs) are the new hotness. If sticking to PNA, who has experience for a good supplier for the EU?
I just want a general (though accurate) estimate for telomere length per patient. Should I get TelG or TelG probes, or both and mix them? Also, I want to use a green fluorophore, AF488 is a lot more expensive than FITC, are signals so weak that it merits the extra $$?
Assuming it's best to include a DNA dye to correct for pleudity, I was thinking of using LDS751, but considering the plethora of new dyes out there I'm open for alternatives. Will 7-AAD work for cell cycle analysis, or one the new patent dyes (RedDot2, DyeCycle Ruby)? It needs to be excited by the 488 laser and emit in the red spectrum as not to give too much overlap with my FITC signal. Also as little as possible excitation with the HeNe laser would be nice, as I need that for other stuff.
I've worked with PNA probes and they work well. FITC is quite bright, I wouldn't think that would be a great deal different. I think the AF's big advantage is that they are more stable. There are tons of nucleic acid probes but given that your telomere probe is likely to be significantly dimmer than your total DNA probe I think you may want to consider using a different excitation - ie why not use a more standard DAPI stain? Or pick a far-red stain. Anything that'll avoid spectral overlap. You could also consider using a centromere targeting PNA probe as the control if you want to get really fancy. As a word of caution: PNA probes and the like are great for in situ staining but I really don't know if this is a good approach for a flow cytometry experiment. You may not get a good enough signal to noise ratio to see anything. RNA binds PNA probes and so cells not RNAse treated have higher background. Furthermore, telomere shortening is stochastic so cells often have one or a few very short telomeres while the remaining telomeres are normal in length. Whole cell assays like flow will not capture this. If you are going to look at whole cells between patients really should just use a qPCR approach - these are by far the most common for this type of bulk measurement and far cheaper, easier, and more proven than a flow experiment. I think you might also want to consider microscopy based experiments if you can - the good news is if you have a flow sorter you can look at the same cells by flow and microscopy so you can directly compare the two methods. In any case you definitely want to make sure your staining is robust by microscopy before you attempt the more risky flow experiment.
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