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I am recapping for the exam in transcriptomics and stumbled into a question about microarray. So, our regular workflow is to

  1. extract RNA from cells
  2. generate cDNA from the initial RNA using reverse transcription
  3. transcribe cRNA from cDNA
  4. label cRNA with biotin
  5. fragment biotin labeled cRNA
  6. hybridize to a plate, scan and quantitate

So the question is - since we use RNA for the further quntification steps why should we perform reverse transcription+transcription to produce cRNA while we could just use the initial RNA?

Is the cRNA more stable than RNA? Or we already make labeled cRNA using labeled nucleotides?

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  • $\begingroup$ Could you give a source or at least application for this workflow? It sounds pretty specialised, both the biotinylation and the in-vitro transcription (cRNA) are - as far as I know - not standard procedures. $\endgroup$ – Nicolai Jan 22 at 10:25
  • $\begingroup$ I don't know exactly the applications of this method, the scheme was shown, for example, in this lecture binf.gmu.edu/jafri/binf636/GMU-higgs.pdf in the section about Oligonucleotide Arrays. $\endgroup$ – Polina Novikova Jan 22 at 10:53
  • $\begingroup$ I can't find any mention of biotinylation or in-vitro transcription/cRNA in the slides you linked, they don't seem to have anything to do with the workflow in your question. $\endgroup$ – Nicolai Jan 22 at 12:23
  • $\begingroup$ It's slide #16 and you have a picture in dark blue. $\endgroup$ – Polina Novikova Jan 22 at 13:26
  • $\begingroup$ Ah, only checked the text of the slides, not the figures as well. I tried to answer - but the figure looks like an example that isn't very well explained (the text next to it doesn't clearly correspond to it) $\endgroup$ – Nicolai Jan 22 at 13:45
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After clarification via the comments of the OPs question:

1) The procedure you describe is not for standard RNA microarray experiments, it for olgionucleotide microarray. This type of chip is special because it has multiple probes for each gene to allow detection of special features (i.e. mutations, alternative splicing, ...).

2) The reason for the generation of cDNA with subsequent reverse transcription and biotinylation is not explicitly stated in the source of the OP, but there are some possible advantages:

  • Amplification of material: cDNA can easily be amplified, which would make it easier to analyse samples whit low RNA yield, genes with low expression levels or to analyse the same sample on multiple chips.

  • Sensivity: oligonucleotide arrays may use very short (10-25 bases) probes, for which fluorescence readout may not be sensitive enough. Biotinylation of a given RNA base (by the use of pre-biotinylated NTPs during reverse transcription) can give multiple 'marks' on each fragment, which can be read with out streptavidin coupled signals. Additionally this would allow the signal strength to change in respond to certain SNPs/mutations.

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  • $\begingroup$ Good intro to oligonucleotide microarray but still not clear why we would convert cDNA back to cRNA after biotinylation. $\endgroup$ – Polina Novikova Jan 22 at 15:25
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    $\begingroup$ @PolinaNovikova my guess is that the final cDNA after doing RT-PCR (not the first strand) which is double stranded is transcribed to make a ssRNA which can then be used as a probe. ssDNA would also work but the cDNA synthesis followed by PCR would not produce ssDNA. You can simply use the first strand after RT but the concentration would be less. With a cDNA library, you can produce a lot of RNA using IVT. $\endgroup$ – WYSIWYG Jan 24 at 11:54
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by @WYSIWYG

my guess is that the final cDNA after doing RT-PCR (not the first strand) which is double stranded is transcribed to make a ssRNA which can then be used as a probe. ssDNA would also work but the cDNA synthesis followed by PCR would not produce ssDNA. You can simply use the first strand after RT but the concentration would be less. With a cDNA library, you can produce a lot of RNA using IVT.

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