Screening for cell cycle Associated genes by RNA AI starts with choosing an appropriate knockdown technology mission.Easy. RNA libraries from Sigma Aldridge makes use of the high efficiency and specificity of endo ribonuclease prepared si. A easy RNAs are complex pools of individual SI RNAs, prepared by enzymatic digest of long double stranded RNA, complementary to the target MNA pooling of SI RNAs targeting the same transcript, increases specificity, and prevents the laborious search for efficient and specific individual SI RNAs.
This makes easy RNA especially suitable for large and medium scale loss of function screens. Here we show the steps necessary for performing a large scale RNA eye screen, starting from setting up high throughput transfect to the development of an appropriate assay. This is exemplified by setting up a screen for genes involved in mammalian cell cycle progression.
First, we show how transfection of hilar cells as optimized, followed by statistical evaluation of the assay quality. Finally, a genome scale screen is performed measuring cell cycle distribution after target gene depletion. The hits are selected using the zco statistic and verified by secondary independent easy RNAs.
Furthermore, we outline potential experimental procedures that can be used to follow up the RNA eye screen. Hi, I am Ty from the Max Plank Institute of Molecular Cell Biology and Genetics. And Tristan.
I'm Frank Al, also from the Marx Plunk Institute in Dresden. Today we will show you a procedure for setting up a loss of function screen in mammalian cells utilizing mission easy RNAs from Sigma Aldridge. An essay for measuring cell cycle distribution in a population of tissue culture cells will be used as an example to demonstrate how genes can be linked to biological process using RNAi screening.
So let's get started. Every RNAi screening project starts with a choice of silencing triggers, employed endo ribonuclease prepared si, RNAs, or easy RNAs for short are generated by limited enzymatic digestion of long double stranded RNA using RNAs three. Every easy RNA is verified for identity by sequencing and for purity by caliper lab chip analysis.
Pooling of individual siRNAs all targeting the same transcript increases specificity because every irna in the pool shares the same on target, whereas the off-target effects are diluted out. Therefore, easy RNAs have good silencing efficacy and high target specificity, avoiding off-target effects. Genome scale easy.
RNA libraries are provided by Sigma Aldrich. In 384, well plates with the edge wells empty. Additionally, eight positions in the center of the plate are left empty for controls.
Sub libraries of easy RNAs are provided in 96 well plates with all wells occupied here, the customers can decide on how the easy RNAs will be arrayed in those plates. Individual easy RNAs are provided in single tubes. Transfection of easy RNA requires optimization for different transfection reagents and cell lines.
Here heal our cells cultured following standard procedures and oligo as transfection reagent is used. Use EEG five E-Z-R-N-A-A kinesin motor protein required for bipolar spindle assembly as a positive control and ran luciferase easy RNA as a negative control. In a 384 well plate titrate different amounts of easy RNA, for instance, from one nanogram to 56 nanograms with an increment of five nanograms and increasing amounts of transfection reagent from 0.1 microliters to 0.9 microliters with an increment of 0.05 microliters.
After mixing the RNA and the transfection reagent, add the cells and incubate for 48 hours. When the incubation period has finished, check the cells under a light microscope. Count the cells transfected with eeg.
Five easy RNAs that show a round shape indicative of mitotic. Rest also count the total number of cells transfected with a negative control. Gran vanilla luciferase easy RNA.
The optimal transfection condition shows the least toxicity for vanilla luciferase and the most pronounced phenotype for EEG five. Now that the transfection parameters are optimized, proceed to set up and primary screen. Automated pipetting stations such as Matrix Wellm Mate or Tecan Aquarius used to prepare the primary screens should be placed under lamina flow hood to avoid contamination.
All used components should be sanitized before use, either by autoclaving if applicable or by spraying with 75%ethanol prior to the screen. These instruments should be optimized according to the manufacturer's protocols. Optimize the pipetting accuracy for all used components on the automated pipetting station because pipetting parameters change depending on the type of solution volume and plate type.
This optimization has to be repeated for every step separately. In addition, optimize the wash protocols so that cross-contamination from well to well is excluded when the automated pipetting stations are ready. Transfect cells in 384 well format using the optimized conditions previously determined to make sure that no position effects are observed.
Use easy RNAs for EEG five and vanilla luciferase with an alternating pattern covering the whole plate. Leave all edge wells empty since a common problem when using multi-well plates is enhanced evaporation at the edge wells during incubation time. This leads to different experimental conditions in different wells, which often translates to plate position effects to further minimize evaporation, employee evaporation barriers such as Corning, breathable ceiling tape.
Also make sure that the incubators are kept at high humidity. However, remember that there are the possible sources for position effects such as variation in liquid handling or the readout system such as a plate reader. After incubation for 48 hours, fix the cells with ice cold, 100%ethanol for two hours, then rehydrate in PBS for 15 minutes.
Stain the fixed cells for 25 minutes in PBS containing one microgram per milliliter of DPI and 100 micrograms per milliliter of RNAs. A finally wash three times with PBS and store at four degrees Celsius. Next, measure the intensity of DPI fluorescence by microscopy.
An Olympus scanner system is used here for each. Well examine the cells and determine the relative DPI intensity. Generate a histogram by plotting DPI intensity against cell number based on the histogram.
Evaluate the cell cycle distribution by calculating the percentage of cells with A DNA content two N for G one G zero phase between two N and four N for S-phase four, N for G two MPH phase and larger than four N for aneuploidy or polyploidy. In order to evaluate the homogeneity of the results over the plate, compare the cell cycle distribution between the different wells by comparing the percentage of cells in G two M phase for the EEG five and luciferase transfect if the results differ significantly over the plate, further optimization is required to eliminate position effects. Once satisfied with the consistency of the results, calculate the statistical differences between the positive and negative controls.
Use the Z factor equation shown here to determine the statistical significance of the results. SD stands for standard deviation and AV stands for average. A Z factor between one and 0.5 indicates a statistically significant separation of negative controls and noise from the positive controls.
If such a statistical difference has been established, one can proceed to the primary screen. Prepare 384. Well tissue culture plates for transfection of easy RNAs utilizing the predetermined optimized conditions here.
15 nanograms of easy RNA per well are dissolved in five microliters of TE buffer. At least eight control positions per plate are loaded with suitable positive and negative controls for the biological process. Studied here, EEG five and ranil luciferase.
Easy RNAs are used to avoid position effects. The edge wells are loaded with five microliters of TE buffer only. Next, add five microliters of opti MEM containing 0.2 microliters of oligo per well mix and incubate for 20 minutes at room temperature.
After mixing the RNA with the transfection reagent, add the cell suspension here. 40 microliters of a he R cell suspension are added at 25 cells per microliter concentration equivalent to 1000 cells per well incubate for 72 hours. After the 72 hour incubation.
Measure the DNA content on an automated microscope such as the Olympus scan R as previously shown. Repeat this procedure for all plates of the library for hit selection. Evaluate the DNA content values using Z-score statistics.
Note that the Z-score is not the same as the Z factor Z-score. Provide a statistical measure for significance of sample value in comparison to a negative or mock control. It is calculated following the equation shown here.
Calculate the zco for all the easy RNA samples using the signal for the negative vanilla luciferase control as reference for hit selection, A threshold has to be applied. Typically, a significance criterion for Z greater than two or smaller than minus two is used. However, depending on the studied biological process or the scope of the screen, different thresholds may be applied.
More elaborate mathematical evaluation methods like the Q can also be used for hit selection. Next, the selected hits are subjected to a secondary screen and HIIT validation. A secondary screen of the selected HIIT genes follows the primary screen to eliminate false positives due to experimental variation.
First, verify the selected hits using the same procedure as in the primary screen, except set up the hits with a bigger number of replicates between three and five to allow for a better statistical evaluation. For the verified hits, use the same assay and readout as in the primary screen, but with a secondary non-overlapping easy RNA or another non-overlapping silencing trigger, such as a chemically synthesized si. RNA Ultimately validate the selected genes by cross-species.
RNAI rescue the gold standard for the verification of RNAi phenotypes available to date. In short, a bacterial artificial chromosome abbreviated back encoding an ortho log from a different species of the selected gene is stably transfected into cells. Back constructs preserve a gene in its genomic context and allow for a nearly physiological expression.
RNAi against the endogenous gene will leave the ortho transgene expression unaltered, which rescues the RNAi phenotype. Finally, the validated candidates has studied in more detail to eventually derive mechanistic understanding. In many cases, RNAi can be used in these secondary more elaborate assays as well.
For example, time-lapse microscopy of RNAi phenotypes. Here is an example of a cell cycle screen in which the DNA content of heal R cells were measured after knockdown of more than 16, 000 genes from the human genome. 1, 351 primary hits were identified to be required for cell division.
In the secondary screen, 743 hits were verified with a secondary independent easy RNA trigger using a secondary assay. Knockdowns that resulted in a G two arrest was separated from knockdowns that resulted in mitotic arrest. Finally, across species RNA eye rescue experiment for the gene, Lin 54 was used to validate its role for proper cell division.
Further downstream experiments showed that depletion of Lin 54 altered the expression of many cell cycle genes. We have just shown you how to set up high throughput transfection of mission, easy RNAs, and analysis of cell cycle distribution in the human tissue culture cell line. When doing This procedure, it is important to remember that transfection conditions and assays have to be optimized separately for any cellular system and any biological process.
The use of automated liquid handling systems is recommended but not indispensable, especially for small scale screens. Manual handling is feasible as well. So that's It.Thanks for watching and good luck with your experiments.
