The aim of this procedure is to visualize messenger RNA that is associated with the endoplasmic reticulum or er. This is accomplished by first plating mammalian tissue culture cells on cover slips. The second step of the procedure is to treat the cells with extraction buffer to remove cytoplasmic mRNA not bound to the er.
The next steps are to rapidly fix the cells and stain the mRNA by fluorescent in situ hybridization. The final steps are to image and then quantify the amount of fluorescence associated with the er. Ultimately, results can show how mRNAs are associated with the surface of this organelle under different conditions.
This method can help answer key cell biological questions such as whether mRNAs that encode secretory proteins can be maintained on the surface of the er, independently of ribosomes or protein synthesis. Visual demonstration of this method is critical as it involves a few tricky cellular manipulation steps. Seed the tissue culture cells on acid treated cover slips for at least one day prior to the experiment cost seven and due to OS cells are both suitable choices as they have high secreted protein production and well-defined ERs.
If an exogenous Mr NA is being investigated, transfect the cells one day after seeding and then incubate the cells for an additional 18 hours to allow for MR.NA expression. On the day of the experiment. To achieve high extraction efficiency, the cells should not exceed 80%co fluency on the cover slip to detect whether mRNAs interact with the ER independently of translation cells can be treated with agents that disrupt the interaction between ribosomes and mRNA at these agents or control media to the cells 30 minutes prior to extraction.
In this case, translational inhibitors, pure Mycin and Homo Harrington in or HHT are used. Begin by warming freshly prepared extraction solution containing dig tonin and a working buffer of one XCHO to 37 degrees Celsius. Set a heating block to 40 degrees Celsius and insert an inverted metal block to form a flat working surface.
Moisten the metal block with water, then overlay it with a fresh piece of paraform and smooth out any bubbles using RNAs free gloves and lint-free tissues. Next, for each pair of cover slips, prepare a six well plate for washing and fixing the cells to four wells. Add two milliliters of warmed CHO buffer to the last two wells.
Add two milliliters of the fixation solution. Then store the plate at 37 degrees Celsius until needed. On the heating block, place a 100 microliter drop of the extraction solution for each cover slip of cells to be processed.
Now using a jeweler's forceps, pick up a cell coated cover slip and dip it into the first and second wells containing CHO buffer. This washes off the growth medium. Then quickly bloss off the excess buffer off the backside of the cover slip.
And briefly place the cover slip cell side down on the extraction solution. After 10 seconds, transfer the cover slip cell side up to the fixation buffer where it should stay at least 15 minutes. The cell extraction step is essential to remove all cyop plasticky mRNA that is not ER bound.
In addition, prepare a control cover slip where the cells are fixed without the extraction. Step after fixation. Wash these unex extracted control cells twice with PBS and then permease in PBS and TRITTON X 100.
Although it is not required, dig in extracted cells should also be washed with the same PBS and Triton solution to reduce background fluorescence after the cells have been permeated. Wash the slides twice with PBS to remove the fixative and detergent. Next, wash the slides twice with 60%fide in one-time sodium citrate buffer or SSC.
However, if an oligo DT fish probe is used to stain all of the cells, poly mRNA reduce the level of fide to 25%Next, prepare the hybridization buffer with the fish probe. Dilute the stock fish probe to 0.2 micromolar in the same concentration of fide in hybridization buffer. Now prepare the staining chamber.
Fill the bottom of a 150 millimeter Petri dish with water. Then float a piece of paraform on the water and turn the dish upside down. The para film will adhere to the bottom of the dish as the water escapes.
If there are any air bubbles, remove them with gloves and lint free tissues for each cover slip pipette 100 microliters of the hybridization solution containing the fish Probe onto the para film. Now place the cover slip cell side down onto the solution. Finally incubate the chamber for five to 24 hours in a warm humidified chamber.
Before the fish staining time is over, prepare an RNAs free washing area, wetter level flat surface with water and overlay it with strips of param. Remove any air bubbles with gloves and lin free tissues. When the staining finishes, transfer the chamber to the working area for each cover slip Pipette one milliliter of wash buffer onto the washing area.
Para film to transfer the cover slips First pipette half a milliliter of fish wash buffer near the edge of each cover slip. The liquid will be drawn under the cover slip by capillary action. Next, using forceps, remove the cover slips from the chamber and place them on the drops of wash buffer.
And wait five minutes. Repeat this wash technique three more times. Using fresh one milliliter drops of wash buffer on the wash area.
Meanwhile, clean glass slides with 70%ethanol and dry them with lint-free tissues. Then for each cover slip, apply 25 microliters of DPI mounting solution to a slide Using forceps, pick up a cover slip and dry its backside with a lint-free tissue to remove excess wash buffer. Then transfer the cover slip cell side facing down onto the drop of mounting solution.
The completed slides can be stored at four degrees Celsius until they are imaged cost. Seven cells that were transfected with plasmids containing placental alkaline phosphatase or cytochrome P 4 58 B one were either extracted with tonin and then fixed or directly fixed to determine the percentage of these mRNA that is ER associated. The non-nuclear fluorescence was quantified in both samples and the fraction of ER associated mRNA was calculated for both mRNAs.
About 60%of the cytoplasmic fraction was associated with the ER to monitor the ER association of these transcripts. After ribosome dissociation transfected costs seven cells were treated with control media, pur MYCIN or HHT, and then extracted tonin and fixed mRNA. Staining revealed that the A LPP, but not the CYP eight P one mRNA remained associated with the ER after ribosome disruption.
mRNA quantification supported this observation and importantly showed that the level of nuclear mRNA was consistent in all samples. These data reveal that a subset of ER targeted mRNAs, such as the A LPP transcript, is maintained on the surface of the ER after ribosome disassembly. Using this protocol, we can begin to investigate how various mRNAs localized to distinct subcellular compartments such as the endoplasmic reticule When combined with other assays used to analyze mRNA nuclear export.
This procedure can be used to study the initial targeting of newly synthesized mRNAs to the surface of the er.
