My summer work has unfortunately not yet led any publishable transcript profiles; however I have made tremendous progress towards my ultimate results through success in troubleshooting! Mycobacterium can prove to be stubbornly refractory to extract good-quality RNA from, due to their resistance to lysis. Consequently, the art of RNA extraction and purification has become all too familiar to me through day to day trial, error, and manipulation of published protocols. RNA isolation is the type of procedure that can be extremely finicky, and every scientist who works often with RNA each has his or her own special variations or preferences, which I have come to acquire as well!
I have been trying out protocols from several different manuals and articles which extract RNA through beadmilling and Trizol Reagent. As straightforward as these methods are, the first few attempts produced degraded RNA on a 2% agarose gel…as did the next 25+ samples. This prompted copious questions that pined over each point of the protocol that might needed to have been altered: Was the RNA not kept on ice properly? Was the problem the agarose gel? Were the reagents contaminated with RNases? Were the samples not immediately processed in RNA Stabilizing Reagent? Could the RNA Stabilizing Reagent even penetrate the thick layer of mycolic acids? Was the sample size too small? Too large? The list went on and on… Each day I came in to the lab, picked a new possible solution and made one single alteration or addition each day…until it worked!
Science doesn’t always happen how you want it to. After spending weeks perfecting techniques on obtaining good quality RNA, I noted that I was only achieving a yield of approximately 1ug/sample (appropriate for my sample size), whereas I need AT LEAST 1.5ug! Therefore, to ensure that not only half of my samples yielded enough RNA, my future work with this project will continue onward into the fall with new trials consisting of significantly scaled-up culture sizes.
Within Pace University’s Biology and Health Sciences Department, I have been working as a research student in Dr. Marcy Kelly’s lab where we are elucidating novel chemo-therapeutics for both early and latent infection tuberculosis. Tuberculosis, pronounced a global health emergency in 1992 by the World Health Organization, claims the lives of an estimated 2 billion individuals each year, meanwhile latently infecting over a third of the world’s population. This deadly disease is on the rise, especially in recent years due to its synergy with the HIV epidemic.
Recent work in Dr. Kelly’s lab has shown that an essential molecule of the human immune system, glutathione (GSH), is toxic to early infection in vitro models after five days, but surprisingly, in latent infection models causes an exit from this non-replicative persistent state in as early as four days, stimulating the pathogen to transition back into active infection. My current project is to use genetic profiling strategies to understand the exact mechanisms by which GSH is toxic to active (mid-logarithmic mycobacteria), as well as the targeted molecular pathways through which GSH stimulates growth out of latency. Thus far, I have performed growth studies of Mycobacterium bovis-BCG, a surrogate for tuberculosis studies in BSL-2 facilities, subjected to 0mM and 8mM GSH over the course of five days. During these trials, I have collected samples of specific time points of interest coinciding with the growth stimulation and killing of BCG. These samples, totaling thirty, are then stored in RNA Stabilizing Reagent to preserve gene expression and prevent any degradation.
I have been working this past week to isolate the RNA of each of these samples (done in triplicate). The quantity of the RNA is then assessed by using NanoDrop technology, followed by a general electrophoresis which illustrates the quality. Furthermore, this week I visited the Center of Genomics at the Public Health Research Institute in New Jersey where I met Director Patricia, and together formulated a schedule for the rest of my experiment and data analysis to be guided and performed at their facility. I am extremely excited for the opportunity to work at such an advanced laboratory and alongside the faculty educated in the use of their cutting-edge biotechnology! Generally, scientists send their RNA in for processing on a gene chip and just receive the data in return, however this summer I have the opportunity to learn how to process and analyze my own transcript samples.