The overall goal of this procedure is to identify in cultured cells or in tissue, the RNA molecular partners of a long non-coding RNA of interest. This method can help to decipher the biological functions of a long non-coding RNA by providing new significant knowledge on RNA regulation by long non-coding RNAS. The main advantage of this method is that it is usable for most applications since we have optimized it, first for several long non-coding RNAS and second for both cultured cells and tissue extracts.
Our method is based on the mix of two previous methods, the ChIRP, which stands for Chromatin Isolation by RNA Purifications, and the CHART, which is the Capture Hybridization Analysis of RNA Targets. The specificity of our method is that the oligonucleotide probes were designed using bioinformatics modeling of a secondary structure of a long non-coding RNA and its goal was to identify the RNA targets. Demonstrating the procedure will be two technicians from my team, Benedicte Boyer and Severine Guillen.
After culturing GH4C1 cells according to the text protocol, remove the medium from a cell culture plate and use one volume of PBS to rinse the cells. Add 10 milliliters of 1%PFA in PBS to fix the cells. Then, cross-link the cells under agitation at room temperature for 10 minutes.
Quench the PFA action by adding 1 milliliter of 1.25 molar glycine and agitate the sample at room temperature for five minutes. Then, discard the solution by aspiration and use 10 milliliters of PBS to rinse the plate two times for five minutes each. Next, add one milliliter of PBS, use a cell scraper to collect cells, and transfer them to a centrifuge tube.
Spin the sample at 510 g and four degrees Celsius for five minutes. Then, remove as much supernatant as possible and store the pellets indefinitely at negative 80 degrees Celsius. To carry out tissue cross-linking, submerge five milligrams of freshly-obtained mouse pituitary gland tissue in a 10X volume of 1%PFA in PBS and agitate the sample at room temperature for 10 minutes.
Quench the PFA by adding 0.05 milliliters of 1.25 molar glycine solution and agitate the tissue at room temperature for five minutes. Aspirate the medium and use 0.5 milliliters of PBS to rinse the tissue two times. After removing as much supernatant as possible, store the cross-linked tissue indefinitely at negative 80 degrees Celsius.
Prepare the lysis buffer then to lyse samples without prior thawing. Use the buffer to resuspend samples in approximately one milliliter per 100 milligrams of cell pellet or tissue. Place the lysed samples in a four degree Celsius water bath and start the sonication series optimized according to the text protocol.
Immediately after sonication, centrifuge the samples at 12, 000 g and four degrees Celsius for five minutes. Then, transfer the supernatants into new tubes. The sonication series must be optimized to be efficient enough to reduce the viscosity from the DNA without obtaining other fragmented RNAs.
To perform hybridization on the sonicated samples, add two volumes of hybridization buffer to the tubes and vortex. Transfer 20 microliters of each sample into centrifuge tubes and store at negative 20 degrees Celsius. Add 100 picomoles of biotinylated oligonucleotide probes to each sample.
Then, incubate the samples under moderate agitation on a tube rotator at room temperature for four to six hours. After the incubation, add 50 microliters of magnetic streptavidin beads supplemented with 200 units per milliliter of an RNase inhibitor solution and a 5 microliter per milliliter protease inhibitor cocktail to the samples. Incubate the tubes at room temperature under moderate agitation on the tube rotator overnight.
The following day, carry out RNA isolation by placing the tubes on a magnetic support and discard the supernatant. Then, add 900 microliters of wash buffer to the beads and place the tubes on a rotator at room temperature for five minutes. Then, place the samples on the magnet, discard the buffer, and repeat the wash five times.
After removing the final wash, add 95 microliters of Proteinase K buffer and five microliters of 20 milligrams per milliliter proteinase-K to the sample. Then, on ice, thaw the input samples and add 75 microliters of Proteinase K buffer and five microliters of proteinase-K. Incubate the samples at 50 degrees Celsius for 45 minutes, then at 95 degrees Celsius for 10 minutes.
Chill the samples on ice for three minutes, then place them on the magnet and collect the supernatant containing the RNAs and discard the beads. Use an RNA purification kit to purify the RNAs. Finally, store the RNAs at negative 80 degrees Celsius and carry out RT and qPCR and construct DNA libraries according to the text protocol.
Based on bioinformatics modeling of the lncRNA secondary structure illustrated here, probes were designed against two lncRNAs, rats Neat1 or Malat1 for GH4C1 cultured cells and mouse Neat1 for pituitary tissue extracts. The relative enrichment of Neat1 or Malat1 was calculated for non-specific and specific probes relative to the input samples. This figure show the efficiency of the specific probes to pull down Neat1 in the rat GH4C1 pituitary cell line and in mouse pituitary tissue extracts.
When the protocol for specific oligonucleotides or SO probes was directed to Malat1, one efficient probe was generated along with one inefficient probe that was discarded. Following RNA pull-downs and RT qPCR, the RNAs associated with Neat1 in GH4C1 cell extracts were also associated with Neat1 in pituitary tissue extracts. Indeed, after Neat1 RNA pull-down, Malat1 was found to be targeted by Neat1 both in the GH4C1 cell line and in pituitary mouse tissue extracts.
In addition, Neat1 was significantly enriched after a Malat1 RNA pull-down was performed with the probe in GH4C1 cells. The close relationship identified between the two lncRNAs is consistent with the potential co-regulatory role of Neat1 and Malat1, suggested by Malat1 knock-out mice that display variations in Neat1 RNA expression. Once mastered, this technique enables in two or three days the capture of the RNA targets of a desired long non-coding RNA.
With the bioinformatic approach, it's important to design several anti-sense probes and then experimentally compare their efficiency because probe efficiency may be altered by cell lysate preparations. Following this procedure, high throughput RNA sequencing can be performed in order to provide the whole RNA interactome of a long non-coding RNA of interest. After watching this video, you should have a good understanding of how to capture the RNA partners of a long non-coding RNA using efficient, specific probes that hybridize to available regions of the long non-coding RNA as determined by bioinformatics modeling of its secondary structure.
