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A Note from Lancer Brown, Program Director of Screening
Before we begin the journey into our RT-PCR Primary screening projects, let's first take a minute to breakdown the meaning of hTERT RT-PCR. 'hTERT' is the gene which codes for the human Telomerase Reverse Transcriptase protein subunit of the telomerase complex. It is hTERT expression which controls how much telomerase activity exists in a given cell. The process of hTERT expression refers to the transcription of the hTERT gene into hTERT messenger RNA (mRNA) which can then be translated into hTERT protein. Both mRNA and protein can be used as indicators of gene expression. 'RT-PCR' stands for Reverse-Transcriptase Polymerase Chain Reaction; this is the assay we use to detect mRNA expression. Reverse-Transcriptase is an enzyme that converts RNA to DNA and in our case we use reverse-transcriptase to make a single stranded DNA compliment (cDNA) of hTERT mRNA. Polymerase Chain Reaction (PCR) is a reaction which uses DNA polymerase and sequence specific primers to copy or amplify DNA to the point of detection. We use sequence specific primers and fluorescent probes so we can detect specific DNA sequences (in our case hTERT) in real-time using a thermalcycler with optics that detect fluorescence. Thus, RT-PCR is the process of converting mRNA to cDNA, and then amplifying a specific DNA sequence to the point where probe fluorescence rises above background on the real-time PCR machine, this point is called a Ct (or Cycle Threshold). The amount of hTERT mRNA starting material is inversely proportional to the Ct score on the real-time PCR machine. If mRNA starting material is high (e.g. more mRNA is expressed) the sooner we are able to detect the amplified cDNA, thus we will see a lower Ct for a sample with higher mRNA expression. Any Ct's below a certain value are considered to be of interest for hTERT induction. Ct scores can then be used in combination with a standard dilution of synthetic target (of known quantities) to estimate how many copies of hTERT mRNA (or transcripts) are induced by compound treatment; we refer to this value as the Number of Induced hTERT Transcripts (hTN). The first step in identifying compounds that induce hTERT mRNA expression is our Primary Screen. This project tests a large collection (>312,000 and counting) of randomly selected compounds for hTERT mRNA induction. These compounds are screened at a treatment concentration of 33.33 uM using high-throughput cell-based hTERT RT-PCR and cell viability assays performed in parallel. Considering the random nature of the compounds we are testing, most will be inactive at inducing hTERT mRNA, so it is beneficial to weed out the negative compounds as quickly as possible. To help with this, we combine two separate wells of treated cells resulting in one hTERT RT-PCR assay for every two compounds tested for hTERT mRNA induction; we call this our 'Duplex' method which doubles the screening rate of our Primary Screen. Reactions that are positive for inducing hTERT mRNA are referred to as Actives, however, this brings up the question, which is the active compound? This question is addressed in Hit Confirmation. Hit Confirmation, the second RT-PCR based screening project at Sierra Sciences, takes each set of Active compounds from the Primary Screen and tests them individually, in quadruplicate under the same treatment conditions in which they were tested during the Primary screen. Compounds that are active in at least two out of four replicates are classified as Hits. The Hit compounds are then categorized based on structural similarities, which is the foundation for organizing our Hits into Hit families. The Hit family core structure is then used to search through our existing random compound library for other similar compounds, which are then prioritized into Primary Screening. This approach allows us to screen families of compounds in a method we call the SAR Focused hTERT RT-PCR Primary screen. To date we have screened 187,888 compounds and have discovered 510 Hits which are grouped into 34 distinct families. The information learned about the various families is used to direct SAR studies in a parallel effort. Using this method we can continue to search for new families of compounds that induce telomerase in the Primary Screen while we improve the potency and efficacy of the active families through structure activity relationships. Lancer K. Brown, M.S. |
SIERRA SCIENCES NEWSAt Sierra Sciences, we're always striving to keep people informed and up-to-date on the latest developments in the fields of anti-aging medicine and telomere biology.
Last Tuesday, Dr. Andrews delivered a presentation on telomerase activation to a group of influential New Yorkers at the Roger Smith Hotel. We'd like to thank Joyce Brooks and Gerard McKeon of Black Tie Magazine for coordinating the event, as well as everyone who worked so hard to make the presentation a success! Photos of the event are already online, and can be viewed at http://blacktiemagazine.com/society_2010_january/Sierra_Sciences.htm Last month, we mentioned that Drs. Andrews and Briggs, and Lancer Brown, will be attending the Keystone Symposia conference "New Insights into Healthspan and Diseases of Aging: From Molecular to Functional Senescence" in Tahoe City, CA. The conference will run from January 31, 2010 to February 5, 2010, and will focus on "the molecular and cellular mechanisms by which tissue and organ function deteriorate and homeostasis fails." In addition, next month, Dr. Briggs will attend the American Association for Cancer Research's fourth Special Conference on the role of telomeres and telomerase in cancer research. The conference will be held in Ft. Worth, Texas, from February 27, 2010 to March 2, 2010. It is expected to draw 250 to 300 telomere biologists and cancer researchers from around the world. We're excited about these opportunities to collaborate with top colleagues on ideas about intervention into the biological processes that cause human aging! ANTI-AGING NEWSIt has been long known that taking large doses of Omega-3 fatty acids can help stave off coronary heart disease, and that they have also been associated with cancer prevention, brain health, increased immune function, and other health benefits. But the mechanisms underlying those beneficial effects have never been clear. However, a new study may have shed some light on those mechanisms. Researchers at the University of California, San Diego recently completed a study of outpatients with stable coronary artery disease who had been taking Omega-3 supplements for an average of six years. Interestingly, they found an inverse relationship between levels of Omega-3 in the blood and telomere shortening: the more Omega-3 found in a patient's blood, the less telomere shortening the patient had undergone over those years. The reasons for this are not clear. It is possible that Omega-3 acids could increase the activity of telomerase in human cells and/or reduce the impact of oxidative stress on telomeres; but, it's also possible that anyone taking high levels of Omega-3 supplements is simply likely to be more concerned with their health in general, and probably exercises more, drinks less alcohol, etc. The UCSD researchers say that a double-blind, randomized, placebo-controlled trial will be necessary to confirm any link between omega-3 fatty acids and cellular aging. Still, it would not be unreasonable to draw a tentative inference that telomere length maintenance may be the mechanism underlying the health benefits of Omega-3 acids. If that's the case, it would be another exciting piece of evidence that we're on the right track! The study, "Association of marine omega-3 fatty acid levels with telomeric aging in patients with coronary heart disease," was published in the January 20, 2010 issue of the Journal of the American Medical Association: http://www.ncbi.nlm.nih.gov/pubmed/20085953
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Sierra Sciences - 1/22/2010 - Volume 1, No. 6
The hTERT RT-PCR High-Throughput Screen at Sierra Sciences is a program consisting of multiple screening projects all linked by a common theme; the identification of compounds that induce Telomerase expression. Together, the screening projects are designed to continuously filter compounds based on their ability to consistently induce high levels of hTERT mRNA at low treatment concentrations. The first portion, our Primary hTERT RT-PCR screen, will be outlined today. This screen is designed to identify core compound structures responsible for hTERT mRNA induction. The core structures are then used as scaffolds to design custom compounds with a high probability of activity using Structure Activity Relationships (SAR). The SAR compound sets are not considered random and are screened in a separate, yet similar, series of RT-PCR screening projects.
To that end, we've launched a Twitter page where we will be posting updates on our research, and the research of our colleagues in anti-aging science, in real time. Come follow us at 
We've also launched a YouTube account where you can watch Dr. Andrews' presentations at last year's A4M conference in San Jose, and at the Manhattan Beach project last November. Be sure to subscribe to our channel so you don't miss upcoming videos! The channel can be found at