rtStar™ tRF&tiRNA Pretreatment Kit is designed to remove modifications in tRF & tiRNA and prepare the RNA samples before tRF&tiRNA cDNA library construction of qPCR. The kit removes 3’-aminoacyl and 3’-cP for 3’ adaptor ligation, phosphorylates 5’-OH for 5’-adaptor ligation, and demethylates m1A, m1G, and m3C for efficient cDNA reverse transcription.
• The first and unique tRF&tiRNA pretreatment kit to prepare RNA samples before tRF&tiRNA cDNA library construction of qPCR.
• Essential RNA modification removals and RNA end conversions are included in the kit in a simple workflow.
• High yields with recovery rate up to 60%.
• Magnitudes of increase in tRF&tiRNA detection sensitivity, quantification accuracy, and assay discriminating power with the treated compared with untreated samples.
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rtStar™ tRF&tiRNA Pretreatment Kit
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tRFs and tiRNAs, generated through precise biogenesis processes from tRNA, perform many biological functions as small noncoding RNAs and are associated with many diseases and conditions. Varying with their particular sources and endoribonuclease cleavages, tRFs and tiRNAs contain many distinct internal modifications and modified termini. While some tRFs and tiRNAs may inherit methylation modifications (such as m1A) and aminoacylated termini from their source tRNAs, tiRNAs, generated by angiogenin, often have 5’-OH and 3’-cyclic phosphate (cP) modifications occurred at the cleavage site. Many current standard cDNA library construction methods, particularly those that target small noncoding RNAs, include adaptor ligation steps in which 5’- and/or 3’- oligonucleotide adaptors are ligated to the 5’ and 3’ ends of the RNA molecule. Because 5’-P and 3-OH at the ends of the RNA substrate are required for adaptor ligation reactions, standard cDNA library construction methods are inadequate for tRF and tiRNA. Importantly, post-transcriptional modifications within the tRF and tiRNA sequence, such as m1A and m3C, also greatly interfere with reverse transcription for cDNA synthesis (Figure 1). In order to efficiently and accurately analyze tRF and tiRNA by qPCR methods, these obstacles must be removed.
rtStar™ tRF&tiRNA Pretreatment Kit is designed to prepare the RNA by removing 3’-aminoacyl and 3’-cP ends, 5’-OH end phosphorylation, and demethylation of m1A, m1G, and m3C. The reagent formulations are optimized to achieve the recovery rate up to 60%. The pretreated RNA samples can be used for subsequent RT-qPCR. The kit is applicable for tRF&tiRNAs of various organisms.
Figure 1. Common modifications on tRF&tiRNA interfere with adaptor ligation and cDNA synthesis, which greatly reduce the performance of subsequent cDNA library preparation of qPCR.
Processing by RNase 1 forms tRNA halves and distinct Y RNA fragments in the extracellular environment. Nechooshtan G,et al. Nucleic Acids Research, 2020
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