The overall goal of this assay is to enrich target transcripts for next generation sequencing. This targeted RNA sequencing assay is a multi-step procedure that begins with the depletion of ribosomal RNA from total RNA, followed by chemical fragmentation and synthesis of complementary DNA, preparation of barcoded libraries, hybridization and capture of targeted transcripts, multiplexed sequencing, and ending with data analysis. This video will demonstrate the procedures for hybridization, target capture, and post capture PCR amplification for four samples.
So this method can help answer key questions in molecular biology, such as identifying gene fusions, single nucleotide variations, relative gene expression, and alternative splicing involving targeted transcripts. The main advantage of this technique is that it is customizable for any genes of interest. To begin the procedure for hybridization, remove from the 20 degrees Celsius freezer and thaw on ice the following:custom hydrated probes, Cot-1 DNA, universal blocking oligos, and adapter specific blocking oligos.
In a low binding 1.5 milliliter tube, combine 500 nanograms of DNA samples, 5 microliters of Cot-1 DNA, one microliter of universal blocking oligos, 0.5 microliters of six nucleotide adapter specific blocking oligos, and 0.5 microliters of eight nucleotide adapter specific blocking oligos. Place the sample tube in a vacuum concentrater with the cap open and facing the opposite direction of rotation. Dry the contents at 45 degrees Celsius for 20 minutes, or until complete evaporation of the liquid.
Next, resuspend the dried content with 8.5 microliters of 2x hybridization buffer, 3.4 microliters of hybridization component A, and 1.1 microliter of nuclease-free water. Allow 10 minutes for resuspension, vortexing every 2.5 minutes. Transfer the resuspended material to a 0.2 milliliter PCR tube and incubate in a thermal cycler at 95 degrees Celsius for 10 minutes.
Remove the hybridization sample tube from the thermal cycler and add two microliters of custom probes resuspended at a concentration of 1.5 picomoles per microliter. Incubate the hybridization reaction overnight at 65 degrees Celsius. The next step after multiplexeded hybridization is the capture of targeted RNA using streptavidin coupled paramagnetic beads.
Remove a bottle of beads from four degrees Celsius and equilibrate it at room temperature for 30 minutes. Prepare 1x working solutions of all the required buffers. Aliquot 140 microliters of 1x wash buffer one into a fresh 1.5 milliliter tube.
Heat this aliquotic buffer and the entire amount of 1x stringent buffer at 65 degrees Celsius in a heat block for at least two hours. Aliquot the appropriate amount of beads into a 1.5 milliliter tube. Place in the magnetic separation rack and discard the supernatant.
Add bead wash buffer and vortex for 10 seconds. Place the tube in the magnetic separation rack for two to five minutes or until the supernatant is clear. Discard the supernatant.
Repeat for a total of two washes. After removal of the bead wash buffer, add an equal volume of bead wash buffer as the initial starting volume. Resuspend and transfer to a 0.2 milliliter PCR tube.
Place the tube in the magnetic rack for two to five minutes or until the supernatant is clear. Discard the supernatant. Place the tube with beads in a mini heat block at 65 degrees Celsius.
Transfer the hybridization sample to the tube with beads, pipette up and down 10 times to mix, and incubate at 65 degrees Celsius for 45 minutes. Every 15 minutes, vortex the sample and spin down for four seconds using a fixed-speed tabletop mini centrifuge. When the 45 minute incubation is complete, remove the capture tube from the heat block.
Add 100 microliters of preheated 1x wash buffer one to the tube, then vortex for 10 seconds to mix. Transfer the mixture to a fresh, low-binding 1.5 milliliter tube. Place the tube in the magnetic rack and allow two to five minutes for separation, or until the supernatant is clear.
Discard the supernatant. Add 200 microliters of preheated 1x stringent wash buffer and pipette up and down 10 times to mix. Incubate at 65 degrees Celsius for five minutes.
Place the tube in the magnetic rack and allow two to three minutes for separation. Discard the supernatant. Repeat the stringent wash once for a total of two washes.
Add 200 microliters of room temperature 1x wash buffer one and vortex for two minutes to mix. Place the tube in the magnetic rack, allow two to five minutes for separation, and discard the supernatant. Add 200 microliters of room temperature 1x wash buffer two and vortex for one minute to mix.
Place the tube in the magnetic separation rack for two to five minutes, and then discard the supernatant. Add 200 microliters of room temperature 1x wash buffer three and vortex for 30 seconds to mix. Place the tube in the magnetic separation rack for two to five minutes, and then discard the supernatant.
Lastly, remove the tube from the magnetic separation rack and add 20 microliters of nuclease-free water to resuspend the beads. Mix thoroughly by pipetting up and down 10 times. Prior to setting up the post capture PCR amplification, remove paramagnetic beads from four degrees Celsius and equilibrate at room temperature for 30 minutes.
Remove the 2x hot start PCR ready mix and PCR primer mix from the 20 degrees Celsius freezer, thaw at room temperature, and then place on ice. Prepare a library amplification master mix by combining 27.5 microliters of 2x hot start PCR ready mix, 2.75 microliters of PCR primer one, and 2.75 microliters of PCR primer two. Set up the reaction in a PCR tube by adding 20 microliters of beads plus captured DNA with 30 microliters of the library amplification master mix.
Cap the tube properly and vortex to mix. Centrifuge the tube for four seconds using a fixed-speed mini tabletop centrifuge at six times G.Put the tube into the thermal cycler and initiate the PCR amplification using the following PCR program. An initial denaturation at 98 degrees Celsius for 45 seconds, 10 to 12 cycles of denaturation at 98 degrees Celsius for 15 seconds, annealing at 65 degrees Celsius for 30 seconds, and extension at 72 degrees Celsius for 60 seconds.
One final extension cycle at 75 degrees Celsius for 60 seconds, and hold at four degrees Celsius. When the PCR is done, remove the sample from the thermal cycler. Add 75 microliters of paramagnetic beads, mix well, and incubate at room temperature for 15 minutes.
Place the tube in the magnetic rack at room temperature for two to three minutes, and then remove the supernatant. Wash the beads on the magnet by adding 200 microliters of 80 percent ethanol, incubating for 30 seconds, and then removing the supernatant. Repeat for a total of two washes.
After removing the supernatant from the second ethanol wash, incubate at room temperature for five to ten minutes to allow the beads to dry, but do not over-dry to cracking. Resuspend the beads in 22 microliters of Tris-EDTA pH 8.0 and allow three minutes for elution. Place the sample on the magnet for three to five minutes.
Lastly, transfer 20 microliters of the eluted product to a fresh, low-binding 1.5 milliliter tube, ensuring no beads are carried over. A gene expression comparison between the capture method and non-targeted RNA sequencing in four cancer cell lines revealed that the capture method enriched targeted genes, shown in blue, by 10 to 1000 fold over non-targeted genes, shown in grey. Additionally, the percentage of reads mapping to targeted regions was increased in capture versus non-targeted RNA sequencing libraries.
An assessment of quality control measures showed that capture and non-targeted RNA sequencing perform equally in terms of alignment to the transcriptome and main insert size. Using the capture method, a higher percentage of exonic regions and a lower percentage of intronic regions were sequenced. As expected, non-targeted RNA sequencing generated over 50 fold more total sequencing reads per sample than capture.
Using the capture method, the percentage of ribosomal RNA sequences present in each sample was lower. Finally, comparisons of single nucleotide variants called in overlapping regions of capture and RNA sequencing for each of the four cell lines demonstrate a high concordance of variance between capture and RNA sequencing within the target region. Once mastered, this technique can be done in three to four days if it's performed properly.
While attempting this procedure, it's important to remember to work quickly during the capture of hybridized targets and ensure the reaction stays at 65 degrees centigrade. After its development, this technique's paved the way for researchers in the field of molecular biology to study transcripts of interest in a high-throughput and cost-effective way.
