AQRA sequencing quantitatively maps small RNAs such as microRNAs, tRNAs, and tRNA fragments in virtually any cell or tissue sample. It can be used to map some of the 170 known tRNA modifications, but there are other methods that are more specific for mapping. You can use AQRNA-Seq to discover disease biomarkers in the RNO and explore protein translation mechanisms at the systems level.

Most RNA-Seq methods can't accurately quantify the abundance of individual RNA molecules in a sample. This is caused by both structural properties of the RNA, such as secondary structures, post transcriptional modifications, as well as the biochemistry of library preparation such as ligation biases of a thousand fold induced by the terminal nucleotides on the RNAs. AQRNA-Seq was developed to overcome several technical and biological challenges limiting the accurate quantification of RNA abundance in a sample.

Compared to other matters, a, achieves linearity between recons and copy numbers of RNA molecules, accurate quantify 75%of a reference library compressing 963 microRNAs with twofold accuracy. AQRNA-Seq is unique in providing absolute quantification of small RNAs. This links the sequencing recounts directly to the number of copies of an RNA molecule in the sample.

This level of accuracy is crucial for comparing dozens to hundreds of tRNAs in a sample and it's unlike regular small RNA-Seq, which only permits relative quantification across conditions and samples. We designed AQRNA-Seq for an unmet need and understanding protein translation. We found that cells reprogram dozens of tRNA modifications and the number of copies of the modified tRNAs to allow selective translation of messenger RNAs that are enriched with codons matching those tRNAs.

AQRNA-Seq allows us to quantify changes in the tRNA pool as part of this mechanism. We've used it extensively to validate this new model for protein translation. To begin, prepare 10 milliliters of 2X AlkB reaction buffer and sterilize through a 0.2 micron syringe filter.

In a sterile PCR tube add 20 microliters of the linker one ligated RNAs, 50 microliters of the 2X AlkB reaction buffer, two microliters of the AlkB demethylase, one microliter of RNase inhibitor and 27 microliters of RNase free water to prepare AlkB digestion reaction. Incubate the AlkB digestion mixture at room temperature for two hours to remove post transcriptional methylation from the RNase. For clean phase separation, add 50 microliters of RNase free water into the AlkB reaction, then add 100 microliters of phenol, chloroform and isoamyl alcohol mixture.

Shake the tube for 10 seconds. Centrifuge the AlkB mixture at 16, 000 G for 10 minutes. Transfer approximately 140 microliters of the top aqueous later containing RNAs into a sterile 1.5 milliliter tube.

To remove residual phenol add 100 microliters of chloroform to the extracted RNase and shake the tube. Centrifuge the mixture at 16, 000 G for 10 minutes and transfer approximately 120 microliters of the top aqueous layer into a sterile 1.5 milliliter tube. After reverse transcription reaction, add one microliter of five molar sodium hydroxide into the RNA cDNA hybrid.

Incubate at 93 degrees Celsius for three minutes to hydrolyze the RNA strand of the RNA cDNA hybrid. Then add 0.77 microliters of five molar hydrochloric acid to neutralize the reaction. Prepare a 3%agarose gel in TAE buffer.

Mix one microliter of 6X loading dye into five microliters of PCR product, and load the gel with the mixture. Load five microliters of 50 or 100 base pair DNA ladders into the well before the first sample and the well after the last sample. Run gel electrophoresis at 120 volts and 400 miliamperes for 75 minutes to locate the PCR products.

After electrophoresis, place the gel in a box and fill it with deionized water until the gel is completely immersed. Add 10 microliters of ethidium bromide into the water, wrap the box with foil and stain for 30 minutes on a shaker. Discard the ethidium bromide containing water into a waste bottle placed in a fume hood.

Rinse the gel with deionized water once and discard the water into a waste bottle. After washing the gel with deionized water for 10 minutes, use a gel imager to visualize the bands and acquire a high resolution image of the gel.