The overall goal of selects experiments is to select RNA molecules which bind specifically and avidly to a target protein in RNA Selects. This is achieved by in vitro transcribing RNA from a library of random DNA oligonucleotides, followed by incubation of the transcribed RNA pool with the target protein. As a second step filter binding is used.
This separates the target binding RNA molecules from the unbound RNA pool. Next, the target binding RNA molecules are alluded. Reverse transcribed to D-N-A-P-C-R amplified and transcribed back to RNA.
This RNA pool, which is now enriched for some binding abers is used for binding to the target in the next select cycle. Generally after eight to 20 cycles, highly avid target specific aptamers are selected and characterized. Hello, I'm gal bean in the Department of neurology at UCLA today, far rahimi.
A senior postdoc in my lab is going to demonstrate a process called cellex. We've used this process in the lab to select abers for amyloid beta oligomers. So let's get started.
For read, it's all yours. In this select experiment, homogeneous aggregate free stable protein ligaments made up of amyloid beta protein A beta 40 are prepared using the pickup procedure as described in a previous J video protocol with modifications detailed in the accompanying written protocol. Following cross-linking, clean up the cross-linked ligament mixture using five milliliter desalting columns.
First, place the column on a stand with a beaker below the column outlet. Remove the top cap off the column and remove the outlet plug. Let the storage buffer flow through and collect into the beaker.
Next, equilibrate the column with five resin bed volumes in three milliliter aliquots of 10 millimolar ammonium acetate pH 8.3. After equilibrating the column apply a 0.5 milliliter aliquot of the pickup reaction mixture to the column. Allow the protein mixture to soak into the resin and discard the flow through.
In the beaker, place the first collection tube below the column outlet. Add 0.5 milliliters of the acetate buffer to the column and collect the first 0.5 milliliter fraction flowing through. Repeat the elution to collective to 8.5 milliliter fractions in the corresponding tubes.
The samples are characterized by SDS page and absorbance. Combine the fractions with the highest protein content and gently mix using a pipette as described in the written protocol. Keep a 10 microliter aliquot for amino acid analysis and store the rest.
Next, prepare the RNA pool. Start by amplifying the synthetic single-stranded DNA library by PCR as described in the accompanying written protocol. Purify the amplified DNA product and verify the amount of DNA by 2%agros gel electrophoresis.
Generally the concentration and yield of DNA are 160 plus or minus 10 nanograms per microliter in 50 microliters per tube. Next in vitro transcribe the DNA in the presence of radioactive CTP overnight, followed by digestion of the amplified DNA template by DNA for four hours to generate a P 32 labeled RNA pool as detailed in the accompanying written protocol. Although the manufacturer Promega recommends a 15 minute incubation with DNAs, we have found in our experiments that a four hour incubation results in complete removal all of DNA template After the incubation with d ns.
Extract the RNA by adding one volume of citrate saturated phenyl chloroform, isoamyl alcohol vortex for about one minute and centrifuge at 16, 000 Gs for two minutes at room temperature after centrifugation transfer the upper aqueous phase to a fresh tube. Using a micro pipette add one volume of chloroform isoamyl alcohol vortex for one minute and centrifuge. As before, remove the bottom phase by aspiration using a micro pipette.
Note that in this step it is easier to remove the bottom phase rather than the supinate. The bottom phase is discarded to waste. Next precipitate the RNA add 0.1 volume equivalent of three molar sodium acetate and one volume equivalent of isopropanol.
Mix and place in a minus 20 degrees Celsius freezer for 15 minutes. After 15 minutes, spin aspirate the supinate and wash the RNA pellet with 0.5 milliliters of 70%ethanol after washing with ethanol centrifuge and discard the ethanol. Dry the RNA pellet at 37 degrees Celsius for five minutes in a heating block, dissolve the RNA sample in 150 millimolar STE buffer pH 8.0 to a volume identical to that of the in vitro transcription reaction.
Heat the tube containing the RNA product at 70 degrees Celsius for 10 minutes in a heating block and vortex. To facilitate RNA dissolution centrifuge at top speed for one minute at room temperature, keep a one microliter Eloqua of RNA for scintillation counting and TPE urea poly acrylamide gel electrophoresis. After dissolution of RNA, remove unincorporated nucleotides using G 50 columns, following the manufacturer's instructions.
Remember to keep one microliter of the G 50 purified RNA for scintillation counting and denaturing gel electrophoresis. Use the two one microliter RNA aliquots from the RNA precipitation and G 50 desalting steps for scintillation counting. Calculate percent label incorporation after scintillation counting and calculation of percent incorporation, calculate the yield of RNA and specific activity of RNA.
Finally examine the RNA before and after G 50 purification by running the samples on a 6%TBE urea poly acrylamide gel and visualizing the RNA by auto radiography as described in the accompanying written protocol. If satisfied with the quality of the labeled product, proceed to mix the RNA library with the protein sample for selects. Incubate the RNA stock at 90 degrees Celsius for 10 minutes for denaturation and then at room temperature for 10 minutes for slow re naturation.
While the RNA is incubating dissolve one protein tube in eight microliters of 60 millimolar sodium hydroxide plus 36 microliters of nucleus free deionized water. Sonicate the mixture for one minute and add 36 microliters of two XRNA binding buffer. For the negative control.
Combine RNA with 20 microliters of 10 XRNA binding buffer and make up the volume to 200 microliters by adding nucleus free water. For the binding reaction, combine 20 microliters of protein and RNA mix with 20 microliters of 10 XRNA binding buffer and make up the volume to 200 microliters. With nuclease free water.
Incubate the tubes for 30 minutes at room temperature while the samples are incubating, prepare the filters and the filter binding setup. First, attach a 125 milliliter sidearm flask to vacuum inlet. Place a pre-cleaned porous glass support for the filter on the sidearm flask.
Next, equilibrate three filter membranes in two to three milliliters of one XRNA binding buffer in a 35 by 10 millimeter Petri dish for 10 to 15 minutes. When the negative control and binding reaction samples have finished incubating centrifuge them at top speed for five minutes. At room temperature, proceed to turn on the vacuum and place the first membrane on the porous glass using a micro pipette drip 0.5 milliliters of RNA binding buffer onto the membrane.
Adjust the vacuum to allow slow flow of each buffer drop through the membrane. This first filter is used only for adjusting the vacuum suction. Next, place the second membrane onto the porous glass and using the same flow rate, apply the negative control onto the membrane.
After applying the sample, wash it four times with 0.5 milliliter aliquots of one XRNA binding buffer. Remove the negative control membrane and place it into a tube for scintillation counting. Replace the porous glass with a pre-cleaned second porous glass support.
Place the third disc onto the porous glass and apply the binding reaction mixture. Wash the filter as just shown the number of washes can be increased in later select cycles. To increase the stringency of the select conditions, remove the filter disc and place it into a 1.6 milliliter tube labeled binding reaction.
Perform scintillation counting for both filters. Calculate the level of the filter bound radioactivity compared to the total amount of radioactivity applied to the membranes. This gives an indication of binding enrichment as the selects progresses.
After scintillation counting, remove the binding reaction filter from the tube and place it in a clean, dry 35 by 10 millimeter Petri dish. Using a clean scalpel and a pair of tweezers, cut the membrane into small pieces. Use the tweezers to transfer the cut pieces back to the scintillation vial.
Add 400 microliters evolution buffer and incubate the tube at 95 degrees Celsius for 10 minutes. At the end of 10 minutes, mixed by vortexing centrifuge the tube at top speed at room temperature aspirate and collect the extraction buffer into a new labeled tube. Measure the remaining radioactivity counts in the tube containing the membrane pieces by scintillation counting To assess the extraction efficiency, repeat the extraction thrice the efficiency after three extractions is usually about 95 to 96%Next, add three to four microliters of glycogen as an RNA co precipitant and extract and precipitate the RNA released from the filter as previously shown except place in a minus 20 degrees Celsius freezer overnight.
After the overnight incubation centrifuge the sample the RNA precipitates into a liquid phase that is barely visible. Aspirate the sate carefully without dislodging this barely visible precipitant phase at the bottom of the tube. Wash the RNA pellet with 0.5 milliliters of 70%ethanol centrifuge for five minutes at top speed at four degrees Celsius and discard the ethanol by aspiration without dislodging the barely visible precipitant phase.
Dissolve the RNA pellet in 50 microliters of STE buffer and proceed to G 50 purification as described for the newly transcribed RNA in the accompanying written protocol. To proceed to the next cycle of selects, reverse transcribe the RNA to DNA followed by PCR amplification as described in the accompanying written protocol. Remember to include a negative control sample to void of the reverse transcriptase.
Check the PCR products on a gel if the negative control lane is empty and the other lanes are as expected. Proceed to transcribe and label the RNA as previously shown for use. In the next cycle of select the pickup generated oligomeric.
A beta 40 protein used here for selects was purified as shown in this video protocol and then examined using absorbance measurements shown by the red trace and SDS page. The protein consistently eludes off the column in fractions three through five and the cross-linking reagent salute after fraction six indicated by increased absorbance in fraction. Seven SDS page shows the typical a beta 40 all ligament distribution.
The integrity of RNA for each select cycle is important for iterative progression of selects. After RNA transcription and labeling, the quality of the labeled RNA is assessed by denaturing gel electrophoresis. Here is a typical profile of an intact labeled RNA product before and after G 50 purification contaminating DNA template from previous select cycles still present after the in vitro transcription reaction could reduce the select sufficiency requiring more cycles.
Aros electrophoresis of the reverse transcription reaction followed by PCR amplification shows the expected DNA product absence of DNA product in the negative control reaction tubes indicates successful removal of the DNA template by DNA's treatment after in vitro transcription. Okay, we have just shown you how to select abers for photo cross-linked covalently stabilized amyloid beta 40 oligomers. When doing this procedure, it's important to remember that both molecules have to have a stable structure in order for them to have high affinity and to remember that both of them have a three dimensional structure in which they interact.
Okay, that's it. Thanks for watching and good luck with your experiments.
