We have developed a defined, reproducible, and longstanding protocol for small RNA sequencing and for analyzing normalized reads using opensource bioinformatics tools. The main advantages of our strategy are that it accurately collects microRNAs through gel purification and that the bioinformatics analysis can be applied regardless of how the microRNAs are collected. High throughput sequencing of microRNAs can reveal insight into disease mechanisms, the processing of microRNAs, and the accurate quantification of gene therapy approaches that deliver small RNAs.
Make sure to work in a RNase free environment and to keep all of the RNA products on ice throughout the procedure. Visualizing the gel cutting steps will help viewers to identify the correct size of the products required for downstream applications. For three prime adaptor ligation, combine 11 microliters of RNA with 1.5 microliters of ATP free 10X T4RNA ligase reaction buffer, one microliter of polyethylene glycol, and 0.5 microliters of three prime linker.
Heat the samples at 95 degrees Celsius on a thermocycler for 30 to 40 seconds, followed by cooling on ice for one minute. Add one microliter of T4RNA ligase two and incubate the reaction at room temperature for two hours. While the samples are incubating, pour a 15%polyacyrilamide gel between 0.8 millimeter separated glass plates in a plastic cast and insert a comb.
When the gel has solidified, add 0.5 5X TBE to the tank and pipette vigorously to wash the wells of residual urea. After pre-running the polyacrylamide gel for 25 minutes at 375 volts, load the samples leaving at least one lane in between each sample. Load 20 microliters of at least two sets of markers in an asymmetrical pattern to keep track of the gel orientation and run the gel at a constant 375 volts.
After 15 minutes, increase to a constant 425 volts for the rest of the run and run the gel for about two hours. At the end of the run, use a plate separator to remove the gel from the glass plates and place the gel on a plastic page protector. Dilute five microliters of ethidium bromide in 500 microliters of distilled water and add the dye onto the marker lanes just above the top light blue marker.
Next, use a clean razor blade to cut the gels from the upper to lower marker in each lane under ultraviolet light and transfer the pieces to a four by four centimeter square of laboratory sealing film. Cut the gel with about three cuts horizontally and two cuts vertically to produce 12 small squares. Add 400 microliters of 0.3 molar sodium chloride onto the sealing film and guide the gel pieces into 1.5 milliliter siliconized tubes.
Agitate the samples on a nutator at four degrees Celsius overnight. The next morning, transfer 400 microliters of each supernatant into new tubes. Add one milliliter of 100%ethanol and one microliter of 15 milligrams per milliliter glycogen co-precipitant per tube.
Then store the tubes at minus 80 degrees Celsius for one hour. For five prime linker ligation, first spin down the thawed samples and resuspend the pellets in water. Next, add 0.5 microliters of 100 micromolar five prime linker, one microliter of T4RNA ligase buffer, one microliter of 10 millimolar ATP, and one microliter of polyethylene glycol to each sample.
Heat the reactions at 90 degrees Celsius for 30 seconds before placing them on ice. Then add one microliter of T4RNA ligase one to each tube and incubate the reactions at room temperature for two hours. For reverse transcription, collect the samples by centrifugation and resuspend the air dried pellet in 8.25 microliters of nuclease free water.
Add 0.5 microliters of 100 micromolar reverse transcriptase primer and five microliters of 2X reverse transcriptase reaction mix from a complementary DNA synthesis kit. After three minutes at 42 degrees Celsius, add 1.5 microliters of 10X reverse transcriptase enzyme to each sample for a 30 minute incubation at 42 degrees Celsius in a thermocycler. After neutralization, prepare a PCR reaction with 29.5 microliters of water, five microliters of 10X taq buffer, one microliter of nucleoside triphosphate, two microliters of 25 micromolar forward primer, two microliters of 25 micromolar reverse primer, 0.5 microliters of taq polymerase, and 10 microliters of the reverse transcribed complementary DNA.
Then run two PCR reactions. For agarose gel purification, prepare a 4%agarose gel with low melting agarose and load 40 microliters or more of the PCR product onto the gel with loading dye and 100 base pair and 25 base pair size markers. After running the gel, cut the band above the 125 base pair band and use a gel extraction kit according to the manufacturer's instructions.
Shake to dissolve the gel band in buffer at room temperature before using the appropriate equipment to quantify the final sequence library. Then use the appropriate opensource bioinformatics tools to analyze the sequence data. In this representative PCR reaction on low melt agarose gel, the acquisition of the correct cloned product compared to the obtained linker linker product and unsaturated versus saturated samples can be observed.
After alignment to human microRNA hairpins, a strong concordance between the microRNA read counts in each replicate can be observed. A total of 306 microRNA species were detected with the greatest number of reads mapping to microRNA 122. It is important to remember to cut the gels at the appropriate size to enable an accurate recovery of the microRNAs.
After sequencing the microRNAs, users can apply a bioinformatics analysis to characterize the distribution of microRNAs within their tissues of interest. This technique has been used to identify how microRNAs are processed and which sets of microRNAs are altered in certain cancers. Be sure to exercise care when handling the ethidium bromide and when looking at the gels under ultraviolet light.
