Hundreds of genes have been identified whose function affects how an organism ages. But many of their relationships are poorly understood. This method can help answer a key question in the aging field.
Namely, what are the genetic inter-relationships between gerA genes? The main advantage of this technique is an increase in throughput, the number of genetic predebations that can be quantitatively measured simultaneously is increased by at least an order of magnitude. Lifespan data, collected with this procedure can be plotted and analyzed using software we provide with this publication.
In addition to provide insight into lifespan, this method can also be applied to other age-related phenotypes, such as stress resistance and changes in health plan. For this procedure, create a fresh stamp of the RNAi sub-library. First, aliquot each well of a 96-well plate, with 200 microliters of LB with ampicillin, using a 12-channel pipetter.
Sterilize a 96-pin plate replicator by sequentially immersing the pins in 50%bleach followed by ultra pure water. Then dip the tips in ethanol, and carefully flame the tips. Next, carefully remove adhesive foil cover of a frozen, 96-well glycerol stock library plate, and gently but firmly grind the tips of the sterilized plate replicator into the wells of the still frozen glycerol stocks.
Then inoculate the cultures and seal the inoculated plate with the permeable membrane. Allow cultures to grow overnight on the bench top. The next day, sterilize the plate replicator, and carefully remove the seal from the overnight culture, and immerse tips of the replicator pins.
Next, inoculate a rectangular LB ampicillin and tetracycline agar plate with the pins. Apply even pressure, and use a small, circular motion to transfer the bacteria without letting the spots get too crowded. Then, incubate the plates overnight at 37 degrees Celsius.
The next day, for every 10 replica plates needed, load each well of a deep-welled 96-well plate with 1.5 milliliters of LB with ampicillin. Next, inoculate the wells with the cultured bacteria. Then, seal the plate with a breathable membrane, culture it for 20 hours with shaking.
Control wells should be scattered across the 96 wells, such that when the 96-well sub-library plate is divided into groups of 24, each division of 24 wells has one of each of the controls. The following day, load 120 microliter aliquots of the culture, to the 24-well plates, using a six-well multichannel pipette, with adjustable tip spacing. Then, dry plates uncovered in a laminar flow hood until all liquid has been absorbed.
Do not let them over dry. Be sure to maintain your focus when aliquoting liquid cultures, to ensure that the proper bacterial culture is put into the proper well in the 24-well plate. Replica set experiments start with the set of independent, 24-well replicate plates, each seeded with the same bacteria RNAi clones.
For lifespan experiments, 15 to 20 L1 animals are added to each plate well, and allowed to grow to the L4 stage. At L4, FUdR is added to all wells across replicates. Later in the same day, a replicate plate can be taken from the set and scored as the first time point.
To score lifespan, liquid is added to the wells of the plate to induce animal movement. Animals that do not start to move are prodded gently with a worm pick. The total number of animals, and the number of dead animals is then recorded.
The plate from the first observation is then discarded. At the next observation time point, another replicate plate is taken from the set, and scored in a similar fashion. This is repeated at the determined scoring interval.
Each time with a new plate from the replicate set. Continue scoring a set of plates until no animals remain alive. First, synchronize a population of animals with the same bleach-based prep method used for the traditional lifespan assay.
Then, using a six-well adjustable spacing pipette, and reagent reservoir, seed 15 to 20 L1 animals into each well of the prepared 24-well plates. While seeding the plates, periodically mix the L1 animals into solution to maintain even distribution. Now, let the animals develop to L4.The timing of L4 development must be known for this protocol.
Periodically monitor the development of the worms. When animals reach L4, add 24 microliters of 50x FUdR to each well, and return the plates to the incubator. Using L4 animals is critical.
If hermaphrodites reach reproductive maturity before adding FUdR, their progeny will survive and eat the food. Conversely, adding FUdR too early leads to ruptured animals. At the desired time points, use one replicate plate to score viability.
To do so, flood the well with M9 solution. Before scoring viability each day, sensor any wells that do not have food, appear contaminated, or have animals with morphological or developmental defects. For each well, record the total animals per well, and check the status of non-moving worms, by gently touching each on the head with a worm pick.
After counting all the non-moving dead in a plate, discard the plate. For this procedure, grow transformed e. Coli overnight in LB with ampicillin.
The next day, concentrate the bacteria by centrifugation at 3, 000 G's for 15 to 20 minutes. Resuspend the bacteria in 1/10 of the starting volume. Next, aliquot 200 microliters of the 10x bacteria on to six-centimeter plates.
Prepare three or four plates per test condition, and include two extra plates in case of contamination. Dry the plates in a laminar flow batch, until all the liquid has been absorbed or evaporated. Once dried, store the plates in a plastic box overnight at room temperature to induce the double-stranded RNA production.
Then, store the plates for up to two weeks at four degrees Celsius. For the animals, collect gravid, adult animals in M9.Wash them twice with M9.Repeating the spin at aspiration. Then, resuspend C.elegans in four milliliters of M9, and add two milliliters of hypochlorite solution.
Now, immediately vortex the worms for three minutes, with periodic, vigorous shaking. Then, under a dissecting microscope, look for a cloud of eggs. If the cloud isn't seen, vortex the worms for an additional 10 to 20 seconds.
Next, quickly wash the eggs with M9, two times, as before. And resuspend the washed eggs in three milliliters M9.Then, transfer the eggs to a new, 15-milliliter tube, and incubate tube at 20 degrees Celsius with gentle rotation, and allow the embryos to hatch overnight. The next day, determine the density of the prepared L1 solution by calculating the average number of L1s per microliter in three, 10-microliter drops.
Load the prepared bacteria seeded plates with 50 L1 animals, each. Set up the plates in a 20 degree Celsuis incubator, until the embryos develop to the L4 stage. Normal development from L1 to L4 takes about 40 hours.
Then, to prevent progeny production, add 160 microliters of 50x FUdR to each plate. Then remove the males, as lifespan is typically measured only with hermaphrodites. Once completed, repack the plates into the 20 degrees Celsuis incubator.
Until all the worms are dead, score their viability on a daily basis. To do so, gently touch each non-moving nematode on the head with a platinum wire or eyelash. Non-reactive animals are dead, and must be removed from the plate.
Take note of any anomalies during this routine. Replica set and traditional methods for assaying C.elegans lifespan produce similar results with wild-type N2 animals. For example, feeding-based RNAi knockdown of mml-1 or mxl-2 significantly decreased normal lifespans.
This was measured by both a traditional lifespan assay, and by the replica set assay. Extended longevity can also be detected. Seen here, RNAi for mdl-1 or mxl-1 significantly increased the lifespans of C.Elegans when measured by either methodology.
Using the replica set method, it is possible to simultaneously quantify changes in lifespans across more than 100 conditions, which would not be reasonable with a more traditional approach. For example, a genome-wide feeding-based RNAi screen identified 159 genes necessary for the increased lifespan conferred by decrease daf-2 insulin-like signaling. The relationship between insulin-like signaling and these genes was further described by measuring the health span of the animals.
Health was indicated by c. Elegans thrashing when suspended in solution. After watching this video, you should have a good understanding of how to use feeding-based RNAi to measure lifespan of C.Elegans.
But RNAi is versatile and can be applied for many different phenotypes. Once mastered, the replica set method allows lifespan to be assessed for over 100 RNAi clones each day, if it is performed properly. Early and late time points can be scored quickly.
All the animals in a well will be either alive and moving, or dead. Scoring takes longest when animals are starting to die. Stagger the set up of independent trials with this in mind.
