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Quantitative analysis of mRNA amplification by in vitro transcription.

Publication ,  Journal Article
Baugh, LR; Hill, AA; Brown, EL; Hunter, CP
Published in: Nucleic acids research
March 2001

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

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Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

March 2001

Volume

29

Issue

5

Start / End Page

E29

Related Subject Headings

  • Transcription, Genetic
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA, Messenger
  • Oligonucleotide Array Sequence Analysis
  • Gene Expression Profiling
  • Developmental Biology
  • DNA, Complementary
  • Caenorhabditis elegans
  • Animals
  • 41 Environmental sciences
 

Citation

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Baugh, L. R., Hill, A. A., Brown, E. L., & Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Research, 29(5), E29. https://doi.org/10.1093/nar/29.5.e29
Baugh, L. R., A. A. Hill, E. L. Brown, and C. P. Hunter. “Quantitative analysis of mRNA amplification by in vitro transcription.Nucleic Acids Research 29, no. 5 (March 2001): E29. https://doi.org/10.1093/nar/29.5.e29.
Baugh LR, Hill AA, Brown EL, Hunter CP. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic acids research. 2001 Mar;29(5):E29.
Baugh, L. R., et al. “Quantitative analysis of mRNA amplification by in vitro transcription.Nucleic Acids Research, vol. 29, no. 5, Mar. 2001, p. E29. Epmc, doi:10.1093/nar/29.5.e29.
Baugh LR, Hill AA, Brown EL, Hunter CP. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic acids research. 2001 Mar;29(5):E29.
Journal cover image

Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

March 2001

Volume

29

Issue

5

Start / End Page

E29

Related Subject Headings

  • Transcription, Genetic
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA, Messenger
  • Oligonucleotide Array Sequence Analysis
  • Gene Expression Profiling
  • Developmental Biology
  • DNA, Complementary
  • Caenorhabditis elegans
  • Animals
  • 41 Environmental sciences