HT Q-FISH: How We Measure Telomeres
Don’t be intimidated by the title! Although it appears to be a random string of consonants it actually stands for High-throughput Telomere Quantification by FISH (which stands of Flourecense In Situ Hybridization). I know it’s a mouthful, but it’s actually one of the most critical tools in further uncovering the secrets of telomeres.
What are telomeres?
In order to understand how to measure telomeres it is critical to understand what they are. In this section I’ll do a breif overview to set up context for the rest of this article. However, if you want to gain a deeper understanding, I recommend checking out my previous article: The Two-Faced Story of Our Cells.
Telomeres are essentially caps at the end of our chromosomes. They contain repeating sequences of DNA up to 15,000 base pairs. Why? To function, our cells divides to replinish our organs. During this cellular division our chromosomes divide as well. To prevent important information from being lost telomeres contain “extra” base pairs to ensure that our genetic code stays the same. Considering that our chromosomes are blurprints for who we are it is critical that their integrity is maintained to prevent diseases and malfunctions within the body. Without telomeres the human species wuld not survive.
However, telomere’s are immortal either. Through mulitple cellular divisions telomeres also shorten. Eventually when a cell can divide no more it either becomes senescence cell — one that simply exists — or goes into apoptosis — cell death. The reason telomeres have become a critical focus in the human longevity community is because longer telomeres means that the cell can survive more divisions which holds off the process of aging. Currently, aging is the number one cause of a plethora of diseases. Without aging, humans can live much healthier and longer lives. Understanding the telomere more could unlock the secrets of human longevity and help solve hundreds of the ailments humans face today!
A Brief Overview of HT Q-FISH
HT Q-FISH is a process that has been specifically developed to measure the length of telomeres which is critical to make conclusions about experiments and make different correlations. It’s especially tedious and time consuming because telomeres are minute parts of the body thus requiring ultra-specific equipment to gain accurate results.
HT Q-FISH measures the mean and median length of telomeres in kilobase pairs, the percentage of short telomeres, and a complete histogram of the telomere length distribution. This method is critical for being able to establish connections between telomere length and human disease because it focuses on especially large samples of data. More specifically, it zeros in on the extremely short telomeres which are the suspects of predisposing people to diseases and other issues (because shorter telomeres → more cell devisions → faster cell death or complacency). This makes sense with other finds as we now know that the shortest telomeres provide critical information about cellular health and chromosome stability, not the average length. After all, it is the weakest link that provides the most information.
In Depth Overview
The first step to starting the HT Q-FISH process is to use a 96-well plate to house the sample. A 96-well plate is absolutely critical to to this process because the number of cells required to start this analysis is 0.01x10⁶, in other words 10,000 cells. The type of cells used are also extremely important. The HT Q-FISH method requires cells to be interphase which are cells in the resting phase, aka when they aren’t dividing but not senescence.
Additionally cell plating and incubation help preserve the temperature, humidity, and oxygen/carbon-dioxide levels. This period is critical to the remainder of the study as telomeres need to be stored in optimal conditions.
Lastly, while cells are within the well-plate, scientists carry out a process called methanol: acetic fixation. While that can sound rather intimidating it serves a simple function. Methanol and acetic acid help enlarge the contact of the nuclei with the slide surface. This increases the nuclear diameter and flattens the nucleous out making the cells, and subsequently, the telomeres much more clear. Given how small telomeres are, this is the best way to increase clarity which allows more smooth image processing later on.
This next section is when telomeric-PNA hybridization occurs. PNA stands for peptide-nucleic acid whose primary function is to probe. In a way PNA functions the same way a detective would. On a case, detectives have to be extremely meticulous, looking for repeating patterns to make conclusions. Similarly, PNA probes the cells within the wellplate to find repeating bases, specifically TTAGGG repeats, which signify telomeres. However, it’s goal isn’t to find just one telomere but to locate all of them within the 96-well plate.
Once the PNA has located the RNA (which are in chromosomes that telomeres cap) they will then bind to the structure. The reason this is possible is that RNA’s are negatively charged whereas PNA’s a nuetrally charged. Once the period of destabilization that occurs during hybridization process is complete they are bound together tightly.
Then, Cy3 is deployed which is an abbrevation for Cynanine3. The reason it’s important is because it’s a flourescent dye that’s usually a green, yellow, and orange hybrid. it’s bright colors make it the perfect cell tracing agent and it doesn’t easily fade either making it perfect for highlighting telomeres. However it is critical that Cy3 is used. Not all fluorescence can survive the 85⁰ C temperatures, humidity, dehydration, etc. that telomeres need while they are stored.
This third step is a continuiation of the previous one (more staining occurs). DAPI which is the short form of 4', 6-diamidino-2-phenylindole (I think I’ll just stick to DAPI) is a different type of flourescent dye that is known for it’s bright blue color. It’s specifically made for DNA which is what makes it the perfect candidate for telomere highlighting. This double staning technique helps illuminate different areas of the telomeres that the Cy3 dye may not have caught providing researchers with a more holistic yet quanitifable picture.
Once placed through the HT Q-FISH bioimaging scanner, a plethora of data is made available. One of the specialities of HT Q-FISH that isn’t present in other telomere length scanners is that these telomeres can be sourced from any part of the body. There are no restrictions for cell type necessary and this includes lymphoid cells. This is especially amazing becaus lymphoid cells contain white blood cells that provide immune responses which usually don’t bode well under this type of imaging and analysis. Additionally, as mentioned before, since HT Q-FISH makes room for both Cy3 dye and DAPI different areas of telomeres are highlighted allowing researchers to gain an extra layer of insights (such as tracking cell lineage and making telomere comparisons across different parts of the body).
Not to mention the data that comes from this process is every researcher’s dream. Both Cy3 and DAPI dyes are help quanitfy telomeres, providing an uber detailed database of of how much dye is present, the length of the telomere, which well it belonged to, and additional important markers. This function is a boon especially for researchers looking into cancer and aging. They can then pinpoint the shortest telomeres of the sample and run further tests or curate qualitative data to make conclusions. Along with that, all of the telomere lengths are displayed on a histogram allowing scientists to get a great understanding of the spread of data. And lastly, HT Q-FISH can also handle metaphase cells which is when cells are in the second stage of cell division allowing scientists to gather data allowing them to compare stable cells to dividing cells.
Drawbacks of HT Q-FISH
Despite how groundbreaking HT Q-FISH has been it also has some steep drawbacks to consider. One being that it is extremely time consuming and a labor intensive. It requires very expensive equipment and weeks of time together to run tests, leaving it isolated only to research labs. Not to mention, while HT Q-FISH does make a treasure trove of data available the technology has not been streamlined enough for the machine to draw conclusions. The actual interpretation, cross examination, and conclusions drawn are fully in the hands of people. While this is true in most research cases, given the plethora of data that needs to be sifted this is a particularly grueling shortcoming. The hundreds of thousands of dollars in equipment, countless hours gathering samples, running tests, rerunning tests, and organization of outcomes makes human longevity a very demanding field. The payoffs are astronomical, not only for the scientist but all of humanity, but the cost to get there is equally large.
Final Thoughts
Before you leave I’d like to leave you with some closing thoughts and reflections.
What started out as an irritating mouthful of letters has turned into one of the most fascinating and fulfilling expereinces I’ve ever had. HT Q-FISH has definitely changed the way I look at human longevity, telomeres, and research as a whole. It is clear that there is so much potential in this field and much knowledge to be gained. But even more so, HT Q-FISH may be one of the many methods we need to finally get to the core of the telomere and find out what other secrets it may be hiding. While we are a long ways off from living till 500, we are certainly getting closer to a future where people live much healthier and longer lives. Science is not simply researchers hunched over petri dished in a secluded lab; science is also part of the human condition driven by curiosity and the urge to understand. HT Q-FISH may very well be one of the most technologically advanced keys we’ve had to the mystery we call life.
