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SE-lncRNA Research

Efficient labeling method to achieve high sensitivity and high accuracy of SE-lncRNA quantification

 

Super-enhancer lncRNAs are generally labile and short in half-lives. They can function at low copy numbers per cell to activate the target genes by cis-mechanism. For example, LncRNA-HOTTIP can activate the HOXA gene cluster with an average of less than one copy per cell. 

To accurately detect and quantify the transient, low level SE-lncRNAs, Arraystar scientists have developed an efficient and robust linear amplification method to generate ample fluorophore-labeled cRNA for both polyadenylated and non-polyadenylated transcripts. In this method, an optimized mixture of oligo(dT) and random primers, each containing a T7 polymerase promoter, is annealed to the RNA. The cDNA is synthesized by reverse transcription followed by 5’ adapter annealing and PCR amplification. Finally, cyanine 3- or cyanine 5-labeled cRNA is synthesized by in vitro transcription from the T7 promoter by T7 RNA polymerase (Figure 1).

The labeling procedure greatly increases the cRNA yield from low-abundance or degraded RNA molecules by more than 100-fold over conventional methods.

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Figure 1. Arraystar’s cRNA Labeling Procedure. (1) First strand cDNA synthesis and 5’ adapter annealing. RNA is primed by a mixture of oligo(dT) and random primers containing a T7 polymerase promoter and reverse transcribed into first strand cDNA. 5’-adapter is annealed to the 3’-end of the first strand cDNA during the reverse transcription. (2) PCR amplification. Double strand cDNA is amplified using the RT primer and 5’-Adapter sequences by low cycles of PCR. (3) In vitro transcription and labeling of antisense RNA (aRNA). Fluorescently labeled antisense RNAs are synthesized by in vitro T7 polymerase transcription from the T7 promoter using a Cy3- or Cy5-nucleotide substrate. The linear amplification reserves the native transcript levels and copies along the entire length of the transcript without 3’ bias.

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