To map the binding sites of ribo nuclear proteins on pre mRNAs start by tiling across the designated region of the prem NNA of interest in 30 nucleotide windows and synthesize the tiled oligos onto an array. The oligos are boiled off the array and coun precipitated with an RNP of interest. The total starting pool is labeled with SCI three and the bound pool is labeled with SCI five, and both are applied to the detection array.
The array is then scanned and the feature extraction software is used to interpret the results.Salts. Hi, I'm Kate Watkins from the laboratory of Will Fair brother in the MCB Department at Brown University. Today I will show you a rapid and inexpensive procedure for determining the precise location of an RNA protein complex on prem RA in the lab, rere synthesize prem A as a pool of overlapping oligonucleotides.
This pool can then be separated into a bound and unbound fraction by co immunoprecipitation and then analyzed by a microarray. While this procedure has many applications, we use it to study multiple splicing factors and their role and alternative splicing. So let's get started.
To begin this procedure, use the uc SC genome browser to download particular genes, splice junctions, or other areas of interest from which to design the prem A pool. Select the windows of interest, then tile across them computationally with a 30 nucleotide read length and a 20 nucleotide overlap so that each oligo is shifted 10 nucleotides from the prior one. Now that the 30 nucleotide long oligos are defined flank each with universal primer sequences.
Submit the oligos to Agilent for printing on a custom oligonucleotide microarray. Once the microarray arrives, begin to recover the DNA oligos from its surface by placing the array cover slide in an array hybridization chamber and gently pipetting 500 microliters of deionized water onto the slide. Place the microarray slide face down on top of the cover slide so that the labels on each slide face each other and take care to avoid air bubbles that can disrupt the oligo recovery process.
Close the hybridization chamber and rotate it overnight at 99 degrees Celsius in a hybridization oven The following day, carefully remove the array from the chamber and draw off the water, which now contains the oligos liberated from the array surface. Place the pool of released oligo in a 1.5 milliliter einor tube and sonicate to break up potential clumps at 50%amplitude three times for three to five second pulses each time. Next, amplify the pool by low cycle PCR using universal primers with a T promoter appended to their end.
The number of required cycles may vary depending on the efficiency of oligo recovery. Avoid over amplifying, which is visible as a smeared band on an acrylamide gel. Finally, use the mega short script kit from Amon to transcribe RNA.
Terminate the transcription reaction by phenyl chloroform extraction of the RNA, followed by ethanol precipitation using sodium acetate and also glyco blue as a carrier. Resus suspend the RNA pellet in 105 microliters of TE buffer. To accurately determine the concentration of the RNA.
Use ribo green to generate a standard curve. Then measure using a fluorescence plate reader. The RNA pool is now ready for co immunoprecipitation to begin the pull down.
First, prepare the magnetic beads by combining protein A and protein G dyna beads in a one-to-one ratio to a final volume of 50 microliters per reaction. Concentrate the beads in one place using a magnetic separation stand in order to remove the supernatant. Then wash twice with an equal volume of cold one XPBS after the second wash.
Resus, suspend the beads in an equal volume of cold one XPBS and add two nanograms of the antibody perion. This amount may vary depending on the antibody, so experimentation may be necessary to find the optimal conditions to bind the antibody to the beads. Incubate the mixture overnight at four degrees Celsius on a rotating platform.
In the morning, add two micrograms per reaction of sonicated yeast total RNA to block nonspecific binding and rotate for an additional 30 minutes at four degrees Celsius. Once the blocking step is done, again, use the magnetic rack to remove the supernatant. Then wash the beads twice with cold one XPBS leaving the PBS from the second wash in the tube aliquot 50 microliters of the bead mixture into new tubes.
One tube for each co IP reaction. The beads are now ready for the co ip. Next, prepare the master mix by combining the RNA oligo pool with nuclear cell extract.
As the RNP source, take a 50 microliter aliquot of the master mix as a total sample protection Primers, which are RNA versions of the universal primers can be added to prevent protein binding to the primer sequence or introducing secondary structure to the RNA. To start the co immunoprecipitation, remove the supernatant from the bead aliquots and re suspend the beads in 120 microliters of the remaining master mix. Rock the reactions at four degrees Celsius for one to two hours.
At the end of the incubation, a small Eloqua, including the beads, may be removed from each co ip. For additional testing of binding efficiency or specificity, place the remainder of the reactions on the magnet and remove the supernatant. Following with one to two washes with cold one XPDS.
The RNA bound to the antibody on the beads through the RNP is the IP sample. Re suspend the IP beads in 50 microliters of 1%SDS in 0.1 XTE buffer and heat the samples at 65 degrees Celsius for 15 minutes. To elute the RNA, remove the supernatant, which now contains the RNA that was previously bound to the beads and clean by phenyl chloroform extraction, followed by an ethanol precipitation, re suspend the pellet in 50 microliters of 0.1 XT buffer.
Next, reverse transcribe the samples using the reverse universal primers. Then PCR amplify the samples with the universal primers, one of which should contain a T seven promoter. The number of cycles required to amplify the pool will depend on the efficiency of the co ip.
Using the mega shorts script kit from ambion, transcribe the samples again, but this time add two microliters of amino allele UTP instead of the T seven UTP that comes with the kit. The amino allele UTP will be used later on to label the RNA with the side eyes. When transcription is complete.
Phenyl chloroform extract and ethanol precipitate the samples. Do not use glyco blue or ammonium acetate, which may interfere with hybridization of the samples to the array. Resus suspend the pellet in 4.5 microliters of 0.1 molar sodium carbonate.
To label the samples first, add 2.5 microliters of nuclease free water to the total sample. Add three microliters of S3 and to the IP sample at three microliters of SCI five. Incubate in the dark for one hour.
At room temperature, terminate the reaction by adding six microliters of four molar hydroxyl lamine HCL to each sample, mix thoroughly and spin. Briefly incubate for 15 minutes At room temperature, purify the labeled samples by phenyl chloroform extraction, followed by ethanol precipitation. Most of the color is pulled down into the organic phase with the free nucleotides, so the pellets may not be visibly colored.
Resus suspend each pellet in 50 microliters of 0.1 XTE. Follow the Agilent array hybridization kit instructions to prepare the microarray sample with the following exception. Do not fragment the RNA After adding the fragmentation buffer immediately add the hybridization buffer in order to stop fragmentation.
Place the samples on the array and hybridize at 50 degrees Celsius for three hours while the array is rotating. Prepare the following 50 milliliter solutions in 50 milliliter conical tubes. One tube of two excess SC buffer, two tubes of one excess sea buffer and one tube of deionized water.
After the three hour incubation, carefully unclamp the array slide sandwich and place in the two XSSC buffer. Using a pair of tweezers. Gently separate the array from the cover slide.
Gently remove the cover slide, taking care not to touch the array. As the slide is pulled out, close the conical and gently invert for 60 seconds. Using the tweezers, transfer the array to the one XSSC solution.
Cap the tube and gently invert for one minute. Then transfer the array to the second one XSSC solution and again, gently invert for another minute. Lastly, transfer the array to the tube of deionized water and gently invert for 30 seconds.
Dry the array by placing the edges of the slide on a kim wipe. Continue to rotate the array on the wipe, making sure not to disrupt the side containing the oligos. Place the array in an empty conical tube and proceed to scan the array.
Proceed to scan the array. Here is a representative array. Each feature or oligo is colored either green for the total unenriched pool or red for the IP enriched pool.
The signal at each feature is normalized to the total intensity at that channel. The final output is the log ratio of the red channel over the green channel. At each feature on the array, the average score at each genomic coordinate is mapped to the given location.
An illustration of this averaging step is given for three overlapping 30 nucleotide oligos with scores of two, four, and 0.5, where the average enrichment score for each 10 nucleotide window is shown. Here is an example of a binding scheme for the poly perine tracked binding protein PTB at the CLTA gene gene features are given along the bottom and log average enrichment scores for the PTP bound S are represented by dark red vertical bars. Light red bars show areas that are unenriched for PTP binding.
I've just shown you how to quickly and efficiently create an oligonucleotide pool that tiles across regions of interest, such as alternatively sliced axons. We analyze our pools by microarray, but is also possible to sequence them either by illuminous, Lexi sequencing, or more traditional Sanger sequencing. When doing this procedure, it's important to remember that the creation of the pool is open to modification either at the RNA or protein level.
At the RNA level, we can introduce autologous regions, disease mutations, polymorphisms, or random mutations into the oligonucleotide sequence. At the protein level, the RNA binding factor can be modified by phosphorylation or other post-translational modifications. Also, the binding environment can be manipulated to either increase or decrease levels of additional factors that either compete with or interact with the RNA binding protein of interest.
So that's it. Thanks for watching and good luck with your experiments.
