Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

The overall goal of this procedure is to unbiasedly prepare microRNAs for high throughput sequencing. This is accomplished by first preparing or purchasing DNA oligonucleotides that are competent for ligation to the three prime end of the micro RNA. The second step is to ligate the three prime DNA oligonucleotide to the three prime end of the micro RNA.

Next, the ligation product is gel purified and the five prime RNA oligonucleotide linker is ligated to the five prime end of the micro RNA. The final step is to convert the hybrid molecules to CDNA and then amplify the CDNA by polymerase chain reaction. Ultimately, micro RNA cloning and high throughput sequencing is used to show the transcriptome wide profile of microRNAs in a quantitative fashion at individual nucleotide resolution.

The main advantage of our technique over existing techniques such as micro sequencing microray qPCR blot, is that our technique is more reflective of actual level of microray on a transcript wide lab scale at a single nucleotide resolution. While this method can provide insight into microRNA biology, it can also be applied to other high throughput sequencing technologies like HC clip and RNA-Seq. The DNA oligonucleotides for this procedure are designed for three prime ligation reactions and should have a five prime phosphate group, a randomized dinucleotide at the five-prime end and a three-prime cytosine dilute the DNA oligonucleotides to 100 micromolar in nuclease free water.

Assemble the a ventilation reaction by combining these reagents agents 200 pico moles of the three prime linker oligonucleotide, four microliters of five prime DNAA ventilation buffer, four microliters of a TP four microliters of M-T-H-R-N-A, ligase and nuclease free water to a final volume of 40 microliters. Incubate the reaction at 65 degrees Celsius for one hour. Terminate the reaction by heating to 85 degrees Celsius for five minutes.

Precipitate the TED linker by adding 2.5 volumes of 100%ethanol and one third volume of 10 molar ammonium acetate. To remove residual a TP and to prevent unanticipated ligations in future reactions, mix the alcohol, salt and oligonucleotide solution to homogeneity by spinning at full speed in a micro centrifuge at four degrees Celsius for 20 minutes. Wash the pellet in 70%ethanol air dry and resuspend the pellet in the appropriate volume of nuclease free water to yield 50 to 100 micromolar of a ventilated oligonucleotide.

Confirm a ventilation of the oligonucleotide by electrophoresis on an 18%poly acrylamide gel. Pre-run the gel for 30 minutes at 24 volts centimeter of gel width with one XTBE running buffer at room temperature. Once the 30 minutes are up, flush the wells with running buffer.

The DNA oligonucleotides for this procedure are designed for three prime ligation reactions and should have a five-prime phosphate group, a randomized dinucleotide at the five-prime end and a three-prime oxytocin. Dilute the DNA oligonucleotides to 100 micromolar in nuclease free water. Run the gel at 24 volts per centimeter until the bromo phenol blue is three quarters of the way to the bottom of the gel post.

Stain the gel with one x cyber gold solution in one XTBE and visualize on a UV transluminator box. There should be a single nucleotide shift in the size of the DNA oligonucleotide that was subject to a ventilation by M-T-H-R-N-A ligase for the three prime ligation reaction. Assemble the following components on ice in this order.

One microliter of 50%PEG 8, 000 0.5 microliters of 10 XRNA ligase buffer. One microliter of a ventilated three-prime linker, 0.5 microliters of RNAs inhibitor 0.5 to eight micrograms of total RNA 0.5 microliters of T four RNA Ligase two and nuclease free water to a final volume of five microliters. Mix the reaction by pipetting once thoroughly mixed.

Place the reaction in a thermal cycler with a heated lid. Set the block to 16 degrees Celsius and incubate for four hours. Note that the reaction may be scaled up to a final volume of 20 microliters without any loss in ligation efficiency.

Following the three prime ligation, mix the reaction with an equal volume of two XRNA loading buffer heat to 70 degrees Celsius for five minutes and snap cool on ice. Next gel. Purify the sample on a 15%poly acrylamide gel.

Pre-run the gel at 24 volts centimeter of gel width for at least 30 minutes. Turn off the power supply and flush the wells with running buffer. Load the sample and run the gel at the same voltage as the pre-run until the bromo phenol blew has just exited the gel.

The desired product will migrate close to the xylene cyan when the poly acrylamide gel electrophoresis is complete. Place the gel in a clean tray with one x running buffer and one x cyber gold and rock for 15 minutes. Remove the gel from the staining dish and visualize on a UV transluminator box covered with clean plastic wrap.

The ligation product should be around 45 nucleotides in length. Since this ligation product is usually not visible, it is important to include a positive control synthetic RNA sample in the adjacent lane using a clean razor blade exci, the region containing the ligation product. Guided by the positions of the synthetic RNA sample and the size standards.

Place the excise gel fragment in a 0.5 milliliter micro centrifuge tube. Pierce the bottom of the 0.5 milliliter micro centrifuge tube with a 30 gauge needle and place the pierce tube in a second clean low retention, 1.5 milliliter micro centrifuge tube centrifuge for five minutes at about 15, 000 times. G.Visually confirm that the entire gel slice has moved through the hole in the inner tube.

If necessary, use a clean pipette tip to push some gel fragments closer to the hole. Discard the empty 0.5 milliliter tube and add 400 microliters of high salt column buffer to the acrylamide slurry. Rock the tube at four degrees Celsius overnight on the following day.

Transfer the slurry to a 0.22 micron cellulose acetate spin column using a wide bore pipette tip centrifuge for three minutes at room temperature at about 15, 000 times. G to collect all liquid away from the acrylamide gel matrix. Transfer the liquid to a clean low retention 1.5 milliliter tube containing one microliter of a 20 milligram per milliliter glycogen solution.

Add an equal volume of isopropanol and a one 10th volume of sodium acetate and mix the solution by inverting the tube several times. Precipitate in a minus 80 degrees Celsius freezer for 20 minutes. Centrifuge at full speed at four degrees Celsius for 20 minutes.

To pellet the nucleic acid, remove the snat and wash in 70%ethanol air. Dry the pellet for 10 minutes at room temperature and proceed directly to the five prime ligation step. To begin this procedure, add the following components to the nucleic acid pelleted earlier, two microliters of PEG 8, 000 0.5 microliters of RNA Ligase buffer, 0.5 microliters of a TP 0.5 microliters of linker, RNA oligonucleotide and water to a final volume of four microliters.

Mix this sample by pipetting up and down repeatedly for up to five minutes to ensure that the pellet has been completely re solubilized. Once the pellet is in solution, transfer the contents to a clean PCR tube containing one microliter of a one-to-one mixture of RNAs inhibitor and T four RNA ligase one mixed by pipetting up and down. Incubate the reaction in a thermal cycler at 37 degrees Celsius for four hours.

When the reaction is complete, proceed immediately to CD NA synthesis as described in the protocol text following linker ligation to the three prime end of RNA and page analysis of the reactions. Sharp high molecular weight bands are evident in the 100 to 300 nucleotide region of the gel, which indicates that the total RNA sample being used is of high quality. The very bright signal at the 25 nucleotide region of the gel is excess unligated three prime linker synthetic RNA indicates a ligation performed with five picomoles of synthetic micro RNA and a three prime linker, which was gel purified.

Shown here is an auto radiogram of three prime ligation reactions performed with P 32 5 prime and labeled synthetic micro RNA. The number of hours that the reaction was allowed to proceed is indicated at the top, and the numbers at the bottom indicate the amounts ligated as a percentage of the sum of unligated and ligated. This auto radiogram is of five prime ligation reactions performed with P 32 5 prime end labeled synthetic micro RNA three prime linker hybrids.

The numbers at the bottom indicate the amounts ligated as a percentage of the sum of unligated and ligated, and reveal the importance of using a high concentration of PEG 8, 000 small R-N-A-D-N-A. Libraries compatible for high throughput sequencing are subsequently generated by PCR. The proper number of PCR cycles is determined empirically.

In this example, the size of the expected DNA product is 146 base pairs Once mastered, this technique can be performed properly in just two days. While performing this technique, it's important to remember to keep all tips and tubes free of RNAs.