The overall goal of this procedure is to simultaneously measure cellular levels of all transfer RNAs in biological samples by combining in vivo metabolic labeling with macroarray analysis. Transfer RNAs were long considered as housekeeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in response to varying conditions such as cell type, environment, and stress.
The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Comprehending the dynamics of protein synthesis requires methods able to deliver high-quality tRNA profiles. We present here, a reliable and straightforward technique that allows fast and precise quantification of transfer RNA levels in laboratory-grown organisms.
To begin, with an 18-gauge needle, place a single hole in the center of the cap of a sterile 1.5 milliliter micro-centrifuge tube to allow proper aeration. Then, with five microliters of Mycobacterium smegmatis from an overnight starter culture, inoculate 500 microliters of supplemented sterile 7H9 broth. Following standard radioprotection practices, spike the culture media with 20 microcuries per milliliter of phosphorus-32-labeled orthophosphate.
Grow bacteria at 37 degrees Celsius and 1, 200 rpm, in a shaker incubator placed behind a nine millimeter-thick acrylic shield. At midlog phase, transfer the entire culture into a two-milliliter screw-cap tube, and pellet radioactive bacteria at room temperature by centrifuging at 10, 000 times gravity for two minutes. Collect the supernatant containing unincorporated radioactive orthophosphate in an appropriate waste container.
To prepare total RNA, add one milliliter of commercial RNA extraction reagent and approximately 200 microliters of glass beads to the pellet. Securely cap the tube, and disrupt the bacteria in a homogenizer under maximum agitation for two minutes. Centrifuge the sample at 12, 000 times gravity and four degrees Celsius for 10 minutes.
Then, transfer the supernatant to a new two-milliliter tube, and appropriately discard the beads. Add 0.2 milliliters of chloroform, and vigorously shake the tube by hand for 15 seconds. Then, centrifuge the sample at 12, 000 times gravity and four degrees Celsius for 15 minutes.
Collect the aqueous phase of the sample into a new tube by angling the tube at 45 degrees. Avoid drawing any of the interphase or organic layer. Add two microliters of colored precipitant and 0.5 milliliters of 100%isopropanol to the aqueous phase.
Shake the tube vigorously by hand for five seconds and centrifuge again. Remove the supernatant from the tube, and appropriately discard it, as the liquid fraction may contain traces of radioactivity. Then, after air drying the pellet for five minutes, resuspend it in 200 microliters of 2X SSC with 0.1%weight per volume SDS.
Using the Genomic tRNA Database, design DNA probes by first retrieving tRNA sequences or tRNA encoding genes. After trimming conserved three-prime end CCAs if encoded and generating the reverse complement, order probes based on the first 70 nucleotides. To carry out array printing, thaw the 96-well plate at room temperature.
With a diamond pen, label amine-coated glass slides. Then, place the slides into an indexing unit. Following the grid placements, carefully dip the replicator pins in the wells.
Using minimal pressure, gently print the array on the glass slides. Continue printing without cleaning the arrayer until finished with block A.It takes some practice to be able to print consistently. We recommend printing no more than eight to 10 arrays per session.
Count 10 to 15 minutes per array. It is easy to lose track of printing pattern sequence, so commit your entire attention to the task and turn off all distractions. When ready to move on to the next block, dip the replicator in 5%bleach and gently shake.
Press the replicator into absorbent paper. Then, dip the replicator in distilled water, gently shake, and press into paper. Repeat this step once.
Next, dip the replicator in isopropanol, gently shake, and press it into the paper. Dry the replicator on the fan for about 20 seconds before moving on to the next block of the 96-well plate. Continue to the desired number of prints.
Then, allow the slides to dry. Place the dried slides face up on a clean surface in a 254-nanometer UV crosslinker. Set the energy level to 999, 990 microjoules per centimeter squared, and then press start.
Transfer the arrays into 450 milliliters of blocking solution, and incubate them at room temperature on a magnetic stirrer, under slow agitation overnight. Wash the slides in 500 milliliters of distilled water at room temperature, two times for five minutes each. Dry the slides by centrifuging in a microarray centrifuge at room temperature for 10 seconds.
Store the arrays in a dry and dark place for up to six months. Prior to hybridization, rinse the slides in boiling water and dry them by centrifugation. Place the array inside a hybridization cassette, and load the radiolabeled RNA sample.
Run the samples on an automated hybridization-washing station for maximum reproducibility according to the text protocol. At the end of the run, dry the slides by centrifugation. Wrap slides with thin plastic.
Then, use a Geiger counter on its most sensitive setting to check the slides for radioactive signal. Expose the slides on a storage phosphor screen in an exposure cassette at room temperature for 10 to 90 hours depending on the signal strength. Exposure time that can last a few hours to a few days depends directly on the array signal.
It takes some practice to be able to translate counts on the Geiger counter into exposure time. Scan slide at 50-micrometer resolution using a phosphorimager. Quantify and background-subtract radioactivity intensities at each probe spot using the free ImageJ software upgraded with a microarray profiler.
Generate heat maps according to the text protocol. Shown here, are scanned bacterial, mouse, and human macroarrays. M.smegmatis arrays use only 43 probes instead of 48 used for mouse and human.
As a result, the bacterial array displays 40 empty spots that blend with the background. Three independent biological replicates show that under the tested growth conditions, all M.smegmatis tRNAs are expressed above background level. In this particular experiment, the observed standard deviation for each probe is between 2%Arginine TCT and 22%Cysteine GCA with the median at 5%In addition, this experiment shows that the expression of tRNA isoacceptors is not uniform.
For example, all Alanine isoacceptors are expressed at similar levels, whereas the highest and lowest Arginine accepting tRNAs differ by three-fold. Once mastered, this technique can be done in approximately 24 hours if it is performed properly and if the spot signal is adequate. Our method is applicable to any organisms whose genome is available for probe design.
Model organisms that are grown in vitro are ideal candidates for metabolic labeling. The protocol presented here is optimized for Mycobacterium smegmatis, but it was also successfully used to profile tRNA in E.coli, yeast, mice, and human cultured cells. Our technique is reproducible and specific.
Its large dynamic range and easily adjustable threshold allows profiling low abundant species, such as tRNAs associated with polysomes, simply by prolonging array exposure times.
