In traditional gene expression profiling experiments, RNA labeling is typically initiated by oligo-dT-primed reverse transcription, which cannot generate Cy3- or Cy5-labeled antisense RNAs along the entire length of the transcript without 3' bias. Furthermore, this labeling method excludes a significant fraction (>25%) of RNA molecules that lack a classical poly(A) tail [1, 2].
To solve this problem, Arraystar scientists have developed an efficient, robust, linear amplification method to create ample fluorophore-labeled cRNA for both polyadenylated and non-polyadenylated transcripts. In this method, a mixture of poly dT oligonucleotides and random primers, each containing the T7 polymerase promoter, is annealed to the RNA. Then cDNA is synthesized by the addition of reverse transcriptase followed by RNase H and DNA polymerase. Finally, cyanine 3- or cyanine 5-labeled cRNA is synthesized using T7 RNA polymerase (Figure 1).
This labeling procedure greatly increases the yield of cRNA from low-abundance and degraded RNA molecules by more than 100-fold over conventional methods.
Figure 1. Arraystar's RNA Labeling Procedure
1. First strand cDNA synthesis: RNA is primed using a mixture of oligo(dT) and random primers, each containing the T7 polymerase promoter, to produce single strand cDNA.
2. Second strand cDNA synthesis: T7 template is annealed to the 3' end of the cDNA. Second strand cDNA is synthesized by adding RNase H and DNA polymerase.
3. Labeled antisense RNA (aRNA) synthesis: Cy3- or Cy5-labeled antisense RNAs are synthesized by T7 polymerase-primed in vitro transcription along the entire length of the transcript without 3' bias.
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1. Cheng, J., et al., Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science, 2005. 308(5725): p. 1149-54.
2. Wu, Q., et al., Poly A- transcripts expressed in HeLa cells. PLoS One, 2008. 3(7): p. e2803.