Top : Molecular Biology : RNA : Reverse Transcription (RT) & cDNA Synthesis

Summary: Reverse Transcription (RT reaction) is a process in which single-stranded RNA is reverse transcribed into complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, a primer, dNTPs and an RNase inhibitor. The resulting cDNA can be used in RT-PCR reaction. RT reaction is also called first strand cDNA synthesis. Three types of primers can be used for RT reaction: oligo (dT) primers, random (hexamer) primers and gene specific primers with each having its pros and cons. For a RT reaction, 1-2 micrograms of RNA is typically used. Steps are: 1) RNA is first incubated with a primer at 70 degree to denature RNA secondary structure and then quickly chill on ice to let the primer anneal to the RNA. 2) Other components of RT are added to the reaction including dNTPs, RNase inhibitor, reverse transcriptase and RT buffer. 3) RT reaction is extended at 42 degree for 1 hr. 4) Heat the reaction at 70 degree to inactivate the enzyme. 5) Sometimes removal of the template RNA by treating the RT reaction with RNase H is necessary before using the reaction in RT-PCR.
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Protocols
  • cDNA Synthesis (Lusis Lab, UCLA)
    To convert RNA into cDNA. You will use a protein called Reverse Transcriptase, which is a polymerase that synthesizes DNA from RNA. The reaction requires primers, which can be either oligodT (annealing to polyA tails of mRNA) or random hexamers; the kit used here contains both kinds); nucleotides for DNA synthesis (dNTPs); MgC2 and buffers required by the enzyme, which are all present in the 5x Reaction Mix.
    http://www.genetics.ucla.edu/labs/lusis/cdnasynthe...
    Added: Sat Mar 21 2009, Hits: 17323, Reviews: 0  Write review   Cached
  • cDNA synthesis using superscript II (A. Untergasser)
    A protocol for cDNA synthesis. Takes a little but workes perfectly.
    http://www.untergasser.de/lab/protocols/cdna_synth...
    Added: Sat Jan 31 2009, Hits: 6749, Reviews: 0  Write review   Cached
  • First strand cDNA synthesis PDF file (Practical Approach Online, Oxford University Press)
    Detailed protocol of synthesizing first strand cDNA for RT-PCR or other applications
    http://www.oup.co.uk/pdf/pas/5-2-2.pdf
    Added: Sat Jan 31 2009, Hits: 10127, Reviews: 0  Write review  
  • First Strand Synthesis Protocol with Reverse Transcriptase (NEB)
    General protocol for RT using M-MuLV reverse transcriptase
    http://www.neb.com/nebecomm/products/protocol50.as...
    Added: Sat Jan 31 2009, Hits: 5875, Reviews: 0  Write review   Cached
  • First-strand cDNA Synthesis (Fermentas)
    The protocol should be applicable to any RT reaction using M-MLV reverse transcriptase.
    http://www.fermentas.com/techinfo/modifyingenzymes...
    Added: Sat Jan 31 2009, Hits: 2515, Reviews: 0  Write review   Cached
  • Theory, principles and background info Reverse transcription (Wikipedia)
    An informative introduction to reverse transcriptase and transcription
    http://en.wikipedia.org/wiki/Reverse_transcription
    Added: Sat Jan 31 2009, Hits: 3870, Reviews: 0  Write review   Cached
  • Reverse transcription (Pamela Stanley Lab Wiki)
    http://stanxterm.aecom.yu.edu/wiki/index.php?page=...
    Added: Tue Mar 03 2009, Hits: 4009, Reviews: 0  Write review   Cached
  • Reverse Transcription (RT) of mRNA to cDNA PDF file (Shivdasani Lab, Harvard Medical School)
    http://research.dfci.harvard.edu/shivdasani/protoc...
    Added: Fri Apr 03 2009, Hits: 9454, Reviews: 0  Write review  
  • RT Reaction and RT-PCR (Cowin Lab, New York University Medical Center)
    http://celljunctions.med.nyu.edu/lab/notprivate/me...
    Added: Mon Mar 16 2009, Hits: 5395, Reviews: 0  Write review   Cached