The rtStar™ tRF&tiRNA Pretreatment Kit prepares tRF & tiRNA samples before the cDNA synthesis used for qPCR. The kit generates terminal 3′-OH and 5′-P ends for adapter ligation by removing 3′-cP and phosphorylation of 5′-OH often present in tRF and tiRNA fragments, and internal m1A, m1G, and m3C are demethylated for efficient cDNA reverse transcription.
The complete solution for tRF&tiRNA qPCR sample preparation
The rtStar™ tRF&tiRNA First-Strand cDNA Synthesis Kit converts the pretreated tRF and tiRNA fragments into cDNA for qPCR reaction. The method sequentially ligates the 3′ Adaptor to the 3′ ends of the RNAs and the 5′ Adaptor to the 5′ ends of the RNAs.
The kit efficiently removes internal modifications to ensure the proceeding of reverse transcriptase during cDNA synthesis, efficiently removes terminal modifications to ensure terminal 3′-OH and 5′-P ends suitable for the subsequent adapter ligation, and is capable of distinguishing tRFs & tiRNAs from their precursors.
The kit prepares tRF & tiRNA samples for qPCR cDNA synthesis in two parts: pretreatment generates terminal 3′-OH and 5′-P ends for adapter ligation and demethylates internal m1A, m1G, and m3C; the first-strand cDNA synthesis kit then ligates 3′ and 5′ adaptors and converts the fragments into cDNA.
Standard catalog products
| Product Name | Catalog No. | Size | Price |
|---|---|---|---|
| rtStar™ tRF&tiRNA Pretreatment Kit | AS-FS-005 | 12 reactions | |
| rtStar™ First-Strand cDNA Synthesis Kit (3′ and 5′ adaptor) | AS-FS-003 | 12 reactions |
Key advantages of the rtStar™ tRF&tiRNA kit
Efficient removal of internal modifications (m1A, m1G, m3C) to ensure the proceeding of reverse transcriptase during cDNA synthesis, whereas the regular cDNA synthesis method does not.
Efficient removal of terminal modifications to ensure terminal 3′-OH and 5′-P ends suitable for the subsequent adapter ligation.
Capable of distinguishing tRFs & tiRNAs from their precursors.
Magnitudes of increase in tRF&tiRNA detection sensitivity, quantification accuracy, and assay discriminating power with the treated compared with untreated samples.
Optimized for the nrStar™ tRF&tiRNA PCR Arrays and Pre-designed Primer Sets.
Why tRF&tiRNA sample preparation needs special handling
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. However, the characteristics of tRFs and tiRNAs pose special challenges for their expression profiling.
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 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 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. In order to efficiently and accurately analyze tRF and tiRNA by qPCR methods, these obstacles must be removed first.
From RNA to tRF&tiRNA cDNA in four steps
Purified total RNA containing small RNAs is used as input, with quality and quantity assessment.
The rtStar™ tRF&tiRNA Pretreatment Kit generates terminal 3′-OH and 5′-P ends (removing 3′-cP and phosphorylating 5′-OH) and demethylates internal m1A, m1G, and m3C.
The rtStar™ First-Strand cDNA Synthesis Kit sequentially ligates the 3′ Adaptor to the 3′ ends and the 5′ Adaptor to the 5′ ends of the RNAs, then synthesizes cDNA.
cDNA is used for qPCR with the nrStar™ tRF&tiRNA PCR Arrays or Pre-designed Primer Sets.
Product specifications and documentation
This kit is optimized for the nrStar™ tRF&tiRNA PCR Arrays and Pre-designed Primer Sets, and Arraystar is not able to advise on other applications. Manuals are provided for both the pretreatment kit and the first-strand cDNA synthesis kit.
Where the rtStar™ tRF&tiRNA kit is used
Optimized for the nrStar™ tRF&tiRNA PCR Arrays for high-specificity tRF/tiRNA profiling.
Pairs with rtStar™ Pre-designed tRF&tiRNA Primer Sets for individual tRF/tiRNA quantification.
Enables accurate tRF/tiRNA detection in biofluids, where they are highly enriched.
Supports studies of RNA interference, mRNA stability regulation, apoptosis, and stress response.
Recommended RNA input for rtStar™ tRF&tiRNA kit projects
Common questions about the rtStar™ tRF&tiRNA kit
Featured Client Publications Using rtStar™ tRF&tiRNA Pretreatment
The rtStar™ tRF&tiRNA Pretreatment & First-Strand cDNA Synthesis Kit removes terminal and internal modification barriers for accurate qPCR — request a quote today.