Die Messung der mRNA-Abbau Preise in<em> Saccharomyces cerevisiae</em> Verwenden<em> Rpb1-1</em> Die Stämme

The overall goal of the following experiment is to measure mRNA decay rates in the yeast Saccharomyces visi. This is accomplished by first growing yeast cells to log phase at the permissive temperature. Next, the cells are shifted to a higher temperature in which total mRNA synthesis is inhibited.

Yeast samples are then harvested at predetermined time points for each sample. The total RNA is extracted separated by gel electrophoresis, and transferred to a membrane. Finally, northern hybridization is performed to ascertain any changes in mRNA levels as a function of time.

Ultimately, this protocol can be used to measure mRNA decay rates of individual mRNAs or genome wide in different growth conditions. This method can help answer key questions in the RNA decay field, such as DO RNA decay rates vary in different cellular conditions and genetic backgrounds. Demonstrating this procedure will be Megan Pelli, a graduate student from my lab.

To begin prepare a starter culture by inoculating a single yeast colony into five milliliters of an appropriate selective media for the strain under consideration. If the strain harbors a temperature sensitive allele of RNA polymerase two, grow the cells to saturation overnight at a permissive temperature of 28 degrees Celsius. On the next day, set up a series of larger overnight cultures, each at a total volume of 100 to 150 milliliters and inoculate different amounts of the starter culture into the growth media.

The optimum dilution factor range can be deduced if both the strain doubling time and the total growth time are known. Grow the new cultures overnight at 28 degrees Celsius on the third day and prior to cell harvest. Preheat two tubes containing 15 milliliters of fresh media at 28 degrees Celsius and 60 degrees Celsius respectively.

Also preheat a dedicated water bath at 39 degrees Celsius at the scheduled harvest time. Measure the OD 600 of all cultures. Choose the flask containing cells in log phase growth and transfer its entire content into several sterile 50 milliliter tubes.

Spin down the cells for five minutes in a room, temperature, centrifuge and decant. The supernatant to pool the cells. Begin by adding 15 milliliters of the 28 degrees Celsius preheated media to any tube containing a cell pellet.

After Resus, suspending the pellet transfer its entire content into the next tube. Repeat the serial resus suspension process until all cells have been pooled into a single 50 milliliter.Conical. Transfer the pooled cells into a 250 milliliter flask.

Equilibrate the cell suspension by incubating for five minutes at 28 degrees Celsius. To begin the main experiment, add 15 milliliters of the 60 degrees Celsius. Preheated media to the cells, swirl the flask a few times to mix and immediately place the flask in the 39 degrees Celsius water bath and start a lab timer.

This non permissive temperature shift at 39 degrees Celsius will inactivate RNA polymerase two and suppress genome wide mRNA synthesis. Pipette three milliliters of the culture and aliquot into two sterile 1.5 milliliter micro centrifuge tubes in a mini centrifuge. Spin the tubes down at full speed for 10 seconds and decamp the supernatant immediately freeze the cell pellet in either liquid nitrogen or an ethanol dry ice bath at predetermined time points.

Repeat the three milliliter sample collection and flash freeze processes as many times as desired. Be sure to keep the working flask in the 39 degrees Celsius bath throughout Store the samples at minus 80 degrees Celsius until use. Isolate total RNA from each sample.

Using a standard hot phenol extraction protocol for all samples, load equal amounts of total RNA onto a standard 1.0%aros formaldehyde denaturing gel. Be sure to dedicate at least one outermost lane for a commercial RNA ladder. Run the gel at five volts per centimeter with a one x mop buffer.

After electrophoresis, excise the ladder lane from the gel lengthwise with a razor stain the ladder slab with an AUM, bromide, or cyber gold solution for 30 minutes. At room temperature detain for 30 minutes on a UV trans illumination table. Rest the stained ladder slab and the unstained gel against a transparent ruler.

Visualize the gel with the UV light on record the relative positions of the prominent RNA ladder bands with respect to both the ruler markings and the top or bottom edge of the unstained gel. Next measure and cut a membrane matching the gel dimensions. Transfer the RNA from the unstained gel to the membrane using standard gel transfer techniques after transfer.

Cross-link the RNA onto the membrane either by baking the membrane for one hour at 80 degrees Celsius or by placing the membrane in a UV crosslinker oven. Store the membrane in a plastic bag at minus 20 degrees Celsius until use before continuing with the protocol, set up a radiation safe workbench complete with a polycarbonate shield, Geiger counter and dosimetry Monitor tags on lab coats. To begin thaw the radio label DNA probes on the lab bench preheat the pre hybridization and hybridization buffer at 42 degrees Celsius.

Use approximately 10 milliliters of each buffer for every 100 square centimeters of membrane. Apply the pre hybridization buffer to the membrane and incubate at 42 degrees Celsius for two hours. Afterwards, denature the radio labeled DNA at 95 degrees Celsius for five minutes.

Add the radio labeled DNA directly into the hybridization buffer and mix. Incubate the membrane overnight at 42 degrees Celsius to complete the hybridization process. On the next day, wash the membrane twice with 50 milliliters of room temperature, salt buffer for 15 minutes each.

Then wash the membrane again with 50 milliliters of 65 degrees Celsius detergent buffer for 15 minutes. Finally, seal the membrane in a plastic wrap to preserve moisture and to ensure the membrane does not contaminate the phospho screen. Using a geer counter estimate the radioactivity of the membrane, then place the membrane onto a phospho screen and expose it to the screen.

Scan the phospho screen with a phospho imager Using commercial imaging software. Quantify the amount of target specific mRNA band in each lane by normalizing its intensity against a loading control band within the same lane In a spreadsheet, tabulate the mRNA band intensity for each lane starting from the first sample collected at the zero time point on a semi log graph. Plot the percent RNA remaining versus sample collection time and determine the slope of the best fit line.

Calculate the mRNA half-life for the target gene by dividing negative 0.693 by the slope. When mRNA transcription can be switched off in a temperature sensitive RNA polymerase two mutant one can readily study the dynamics of mRNA degradation by examining mutants defective in nonsense mediated mRNA decay or NMD for short. For instance, in wild type yeast, any accumulation of mRNA species containing a premature termination code on or PTC is quickly suppressed by their targeted degradation by an md.

On the other hand, mutants with a defective NMD pathway will often display a significantly slower degradation rate of the same mRNA type. Thus, the mRNA will have a longer half-life by eliminating all new mRNA synthesis events with a non permissive temperature shift. Two observations can be made from this experiment as expected in a wild type strain with functional NMD pathway and a temperature sensitive RPB one dash one background.

The existence of nonsense containing prem NNA was completely suppressed by normal targeted degradations in an NMD defective mutant strain. However, the targeted degradation rate of the nonsense pre NNA was noticeably slower, which resulted in its increased half-life by quantitating the amount of nonsense containing mRNA. With respect to time with an imaging software, one can deduce the effective half-life of the mRNA from the slopes of the semi log plot of mRNA.

Quantity versus time Once mastered, this technique can be completed in one week if performed properly. After watching this video, you should have a good understanding of how to determine mRNA decay rates and croces.