Structural Biochemistry/Short RNAs

Introduction

Short RNAs play an important role in biochemistry. Short RNAs include transfer RNAs, small nuclear RNAs, micro RNAs, and other ones. In this section, we are gonna talk about how to profile short RNAs using helicos single-molecule sequencing. In order to lengthen the RNAs chain, scientists use the methods of splicing and 3 prime-end processing. And there are many non-protein coding RNAs that can be less than 200nt; they can be called short RNAs.

Materials

In RNA isolation, scientists want to purify sRNA from the total RNA or cultured cells. Hence they use certain materials to do this technique. They are mirVana™ miRNA Isolation Kit, miRNeasy Mini Kit, RNA/DNA kit. The RNA/DNA kit can be used to isolate large amounts of sRNA from the total RNA. Then people use TBE-Urea polyacrylamide gel electrophoresis and overnight elution to obtain the sRNA from each kit. In the process of making cDNA, scientists use Escherichia coli PolyA polymerase and 100 mM CTP substrate. Then they apply the process of reverse transcriptase called ThermoScript. Consequently, Phenol, chloroform, and isoamyl alcohol were used with the 5 M Ammonium acetate. And the cDNA synthesis primer and RNAse A are also used. This sequence of this primer was created by the Integrated DNA Technologies and their sequence is TCG CGA GCG GCC GCG GGG GGG GGG GGrG rGrG. In profiling 3 prime-end sRNAs, the reverse transcriptase called SuperScript III, USER enzyme, dTU-V cDNA synthesis primer with the sequence of TTTTUTTUTUTTTUTTTTUTTTUTTV, RNAse H, and RNAse 1f were used. Overall, the two methods use similar materials in the process. They both used 100 and 70% ethanol, 10 mM dNTPs, AMPure ®, Magnetic stand for 1.5-mL tubes beads, and PCR machine. The RNAse inhibitors were also used in the methods. They are ANTI-RNAse inhibitors or RNAseOUT inhibitors. In sequencing cDNA, scientists use 20 U/ mL Terminal Transferase, dATP, 1 mM Biotin-ddATP, 10 mg/mL Bovine serum albumin, Quant-tT™ OliGreen ® ssDNA Reagent, NanoDrop 3300, HeliScope™ Single Molecule Sequencer, and Helicos ® Flow Cells

Methods

Before beginning to profile short RNAs, scientists have to understand the goals of the experiment. The desired length sRNA must be separated from the long RNAs. They have to understand that only some of sRNAs have the 3 primed polyA tail of the mRNAs that can be used to convert sRNAs to cDNA. Also, using random hexamers can only be used sometimes to convert the short RNAs to cDNAs since the RNAs are too short for the process of conversion to go smoothly. Some RNAs have modifications at their 3 primed and 5 primed ends that would make it harder to make conversion to cDNA. There are two methods in detecting sRNAs. The first method detects sRNAs with the 3 primed -OH. And the second one detects 3 primed-polyA sRNAs. First, the isolation of sRNAs occur using different kits. If only less than 200nt section of sRNAs is needed, the mirVana kit , miRNeasy, or RNA/DNA kit would be useful. The TBEUrea denaturing polyacrylamide gel-electrophoresis can also be used to isolate sNRA for a specific region.

The general method of profiling sRNAs occurs by tailing RNAs with 3 primed polyC. First, they put RNA into a PCR tube with 30μL. The amount of short RNAs used in this step can be around 5ng to 10ng. Then they incubate the tubes in the PCR machine at 85⁰C for 2 minutes, then put the tubes in the ice for another 2 minutes. To the tube, they add 10 mL of 5× E. coli PolyA polymerase buffer; 5 mL of 25 mM MnCl₂, 1 mL of 100 mM CTP, 1 mL of Anti-RNAse or RNAseOUT, and 3 mL of 2 U/ mL E. coli PolyA polymerase. Hence , they mix the solutions and incubate it in PCR machine for 3 hrs at 37⁰C. After that,40μL of water and 10 μL of 5M ammonium acetate were added to the incubated tube. Then they extract the solids twice in the tube with phenol, chloroform, isoamyl acid. Then precipitation of the solution occurs when they add three times the 100% EtOH to the tube at -80⁰C . In the end, they would centrifuge the solution at 4⁰ for 30 mins and wash the precipitation with 70% EtOH, then vaccuum dry the solid. Finally, you have to put the solid in 30.5μL of water.To make cDNA, they first add the 1 mL of 100 mM cDNA synthesis primer to the 30.5μL solution in the previous step. Then they would incubate the solution for 2 minutes at the 70⁰C in the PCR machine. At that temperature, they would add more reagents to the solution like 10 mL of ThermoScript cDNA Synthesis buffer for 5 times ,5 mL of 0.1 M DTT, 2.5 mL of 10 mM dNTPs, and 1 mL of ThermoScript reverse transcriptase. Finally, incubation of the solution is required for 15 mins to inactivate the reverse transcriptase. After the cDNA synthesis process, they purify the cDNA. First, they mix the synthesis cDNA with 1 mL of RNAse A. And the AMPure beads suspension were mixed so that the beads would not be suspended. Then to the mixture of cDNA and RNAse, they added 150 mL of the AMPure beads to incubate the solution at room temperature for 30 minutes. Hence, they collect the beads using the magnetic stand and the solids would be removed from the solution. The solution was washed 2 times with 200 mL of 70% EtOH and the solids would be dried for from 30 to 45 minutes at room temperature. Then they would wash the cDNA two times with 20μL of water. Before beginning to profile short RNAs, scientists have to understand the goals of the experiment. The desired length sRNA must be separated from the long RNAs. They have to understand that only some of sRNAs have the 3 primed polyA tail of the mRNAs that can be used to convert sRNAs to cDNA. Also, using random hexamers can only be used sometimes to convert the short RNAs to cDNAs since the RNAs are too short for the process of conversion to go smoothly. Some RNAs have modifications at their 3 primed and 5 primed ends that would make it harder to make conversion to cDNA.

The second method of profiling sRNAs is to make cDNA by profiling with polyA tails. First, 1 mL of 50 mM dTU-V primer and 1 mL of 10 mM dNTPs are mixed together. Then the solution is incubated for 5 minutes at 65⁰C by using the thermocycler. Then the solution is to be put on the cold aluminum and it was left there for 1 minute. The next step is to add the solution to 2 mL of ten times of SuperScript III reaction buffer, 4 mL of 25 mM MgCl₂, 2 mL of 0.1 M DTT, 1 mL of SuperScript III, and 1 mL of RNAseOut. This mixture will be incubated for 50 minutes at 85⁰C. The next step is to remove the dTU-V primer sequences from the mixture obtained previously. In order to carry this reaction, 1 mL USER enzyme is added to the mixture and the solution is incubated again for 15 minutes at 37⁰C. Then d 1 mL of RNAse H and 1 mL of RNAse are added to the solution. And this solution can be used for the next step which is the purification of cDNA

In this reaction, 180μL of AMPure beads is heated up to room temperature. Then the beads will be added to the solution that was mixed and incubated to make the cDNA solution. The beads are then collected by using the magnetic stand and the solids would be collected too. Then the beads are washed with 500 mL of 70% EtOH two times. The solids would then dried up. Finally, to isolate the cDNA from the beads, 20 mL of nuclease-free water is added to the solids. And the liquid will be removed using the pipet until the cDNA solid is obtained.

Sequencing cDNA

The 3 primed end of the cDNA will be blocked by polyA residues using terminal transferase (TdT). In order to carry out the process, they need to prepare the 3 primed end of the cDNA. First, they got cDNA to be tailed (<10 ng) in 10.8 mL of water. Then 2 mL of 10× TdT buffer and 2 mL of 2.5 mM CoCl₂ are added to the mixture and the mixture is to be incubated for 5 minutes at 95⁰C.4 mL of 50 mM dATP, 0.2 mL of BSA, and 1 mL of TdT are added to the incubated solution. Then the mixture will be incubated in the PCR machine for 60 minutes at 70⁰C. The solution is to be put on ice for 2 minutes. Then 1 mL of 10× TdT buffer, 1 mL of 2.5 mM CoCl₂, 0.5 mL of 200 mM Biotin-ddATP, 6.5 mL of water, and 1 mL of TdT are added to the cold solution. Finally, it will be incubated in the PCR machine for 20 minutes at 70⁰C