The overall goal of this procedure is to assay gene function in a quantitative manner and see elegance at a genome wide scale. This is accomplished by first knocking down the expression of each gene by RNA eye. In 96 well plates.
Next, the relevant test for gene function is performed. In this example, the capacity of sea elegance to respond normally to fungal infection is assessed by analyzing the expression of an inducible fluorescent reporter. Then an automated quantitative analysis is performed using the Coss bio sortt, ultimately, analysis of quantifiable traits such as the expression of a fluorescent reporter gene show subsets of genes that are required for modulating particular pathways, governing development, behavior, or physiology.
This method can help answer key questions in different fields that you see elegance, such as development, neurobiology, as well as host pathogens interactions. We first had the idea for this method when the first large scale RNA eye screens were published by the Oranger lab. It took considerable effort to develop the tools required and to perfect the automation.
I will be demonstrating the procedure together with Olivia ti, a staff scientist and a research assistant from Jonathan Eubanks lab To make 10 to 1296. Well plates begin by preparing 100 milliliters of nematode growth, medium or NGM in deionized water. Please see the written protocol for instructions.
While the medium is still warm, add 100 micrograms per milliliter of ampicillin, 12.5 micrograms per milliliter of tetracycline four millimolar of IPTG and quickly distribute 75 microliters into each well of a 96 well flat bottom plate. Check for and remove any air bubbles present in the wells immediately. Store the plates in a humid chamber at four degrees centigrade.
Next, prepare 96 deep well plates by adding 1.5 milliliters of LB containing ampicillin and tetracycline. To each well add a unique label or barcode. Cover each plate and store at four degrees centigrade if prepared in advance, agitate the 96 well LB stock plate containing the RNAI.
Bacterial clones with ampicillin, tetracycline, and glycerol, and distribute three microliters of each bacterial clone to the corresponding well of a previously prepared 96 deep well plate. Use empty wells for controls covered the 96 deep well plates with an adhesive film and incubate them overnight at 37 degrees centigrade with agitation. Place the used 96 well replicate plate at minus 80 degrees Celsius on the morning following the overnight culture.
Transfer the 96 well NGM plates from four degrees centigrade to a sterile laminar flow cabinet and allow them to warm up and dry for five to 15 minutes. Then add the appropriate label or barcode to each plate. Retrieve the 96 deep well overnight culture plates from the incubator.
Observe the successful growing bacterial cultures centrifuge. The 96 deep well plates for five minutes at 4, 000 RPM. Drain off the supernatant by turning the plate sharply upside down and rapidly dry.
The edges of the plate on a paper towel resuspend the bacterial pellet in the residual liquid by vortexing. Ideally using a dedicated agitator so that each plate receives precisely the same treatment. Transfer five microliters of the Resus suspended bacteria onto the 96 well NGM plates using an eight or 12 channel multi pipette.
Avoid touching the NGM. Let the bacteria dry under a sterile laminar flow cabinet for approximately two hours. Checking regularly to avoid the NGM becoming two dry incubate, the 96 well R-N-A-I-N-G-M plates at 37 degrees centigrade overnight in a humid chamber.
Refer to the written protocol for preparing a synchronized population of worms after cooling down the 96 well R-N-A-I-N-G-M plates at room temperature. Add approximately 100 to 200 worms in two microliters of M nine to each. Well check that each well has worms.
The worms should be swimming. Let the plates dry under a sterile laminar flow cabinet for a maximum of one hour. Check the plates regularly.
The worms should be crawling rather than swimming. Place the plates in a humid chamber at 25 degrees centigrade overnight. After selecting a highly infective batch of Judge Miria spora.
Use M nine Buffer to collect the spores from a nine centimeter plate. Distribute four microliters of spores to each well and check each well for spores. The worms should be swimming.
Set the plates to dry under a sterile laminar flow cabinet for approximately one hour. Check the plates regularly until the worms start crawling. Then place the infected plates in a humid chamber at 25 degrees centigrade overnight.
Check the worms before analysis by rapidly observing them under a fluorescent microscope for successful development for good GFP induction on negative control and for reduced GFP fluorescence on positive control wells. If induction is good, using an eight or 12 channel multi pipette transfer the worms with 100 microliters of 50 millimolar NACL and 0.05%triton to a new labeled or barcoded. 96 well round bottom plate.
Freeze the plates at minus 80 degrees Celsius until analysis on the day of the analysis thaw the frozen plates at room temperature. Once the plates have thawed. Proceed to the automated analysis using the COPA bios.
Sortt the machine treats the plate well by well and analyzes the parameters of each worm, such as their fluorescence. Transcribe the information obtained from COPA bios sortt into an Excel file for verification of data quality. Then store the raw data including optical density, axial length, and fluorescent emissions in a limbs package for subsequent detailed quantitative analyses.
In this experiment, transgenic worms constitutively expressed a P call 12 ds red reporter and an inducible PNLP 29 GFP. Reporter worms were exposed to a control or A-G-F-P-R-N-A eye clone for 48 hours. The panels on the left are uninfected worms, and those on the right are worms 18 hours after infection with D spora.
When the phenotype of interest is the expression of A GFP reporter, gene Wells with the GFP RNAi clone provide a robust control. The COPA bios SORTT generates numerical data for each worm analyzed. These graphs show the average and standard deviation for each well of transgenic worms.
Constitutively expressing a p call 12 DS red reporter and an inducible PNLP 29 GFP reporter after RNAI and infection with diaspora. The sorter analyzes the time of flight or TOF, which corresponds to the size of the worms, the red fluorescence, and the ratio of green fluorescence to TOF. Certain clones provoke growth defects and or affect the expression of P 12 DS red.
Others specifically affect FP expression. For example, this particular clone targets the gene NS Y one previously shown to be important for the regulation of NLP 29. Green and red fluorescence and TOF are measured in arbitrary but constant units.
One of the innovations of this protocol is the use of freezing plates to postpone their analysis. Freezing allows plates to be stored for up to two weeks without substantially altering the results as shown here. Although the absolute values of measured fluorescence may be changed, their relative ratios remain similar.
L four worms carrying PNLP 29 GFP and P call 12 ds red transgenes were either infected with diaspora or not. After 18 hours, each plate was roughly divided in two half was analyzed immediately and half was frozen at minus 80 degrees Celsius for 24 hours before thawing. An analysis values for average size opacity and green and red fluorescence were calculated for each sample.
The graph shows the ratio of the average for infected divided by non-infected samples for the frozen and live samples shown here. From duplicate analyses of a representative plate are the normalized fluorescence ratios for each well, which is the fluorescence mean of a well divided by the trimmed mean of the plate. Once mastered, this protocol can allow a group of two to three people to perform a genome wide screen in two to three months.
The successful execution of this protocol requires a lot of good teamwork. The members of my group invested enormous effort to make protocol that is efficient and relatively easy to carry out.
