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    CDNA Synthesis

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    sama

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    © All Rights Reserved
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    CDNA Synthesis

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    sama
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    Copyright:

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    10 views17 pages

    CDNA Synthesis

    Uploaded by

    sama

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    of 17

    DNase treatment

    &
    RT-PCR
    Quality of RNA

     quality of RNA: The A 260/A280 ratio should be above 1.8.


    The gel electrophoresis pattern should reveal two major bands
    of 28 S and 18 S RNA and no smear from genomic DNA.
     Negative RT-

    1 2
    Before treatment
    After treatment
    1,2
    What is RT PCR?

     Reverse transcription polymerase chain reaction (RT-PCR) is used when the


    starting material is RNA. In this method, RNA is first transcribed into
    complementary DNA (cDNA) by reverse transcriptase from total RNA or
    messenger RNA (mRNA). The cDNA is then used as the template for the PCR or
    QPCR reaction.

    Sample RNA isolation

    RNA RT-PCR

    cDNA
    Difference between RT-PCR and traditional PCR

     Traditional PCR is used to exponentially amplify target DNA


    sequences.

     RT-PCR is used to reverse transcribe mRNA to cDNA and then amplify


    this result using traditional PCR.

     Since mRNA is the message that is sent for translation, RT-PCR can
    give us a measurement of gene expression that PCR cannot.
     The cDNA produced does not contain the introns that DNA does.

     This technique is also used for diagnosing genetic diseases as well as


    studying certain viruses whose genetic information are exclusively
    composed of RNA.
    How to generate cDNA (Complementary DNA) ?

     In order to copy the RNA sequence into complementary DNA sequence,


    we need to use an enzyme called "reverse transcriptase" which is an
    RNA dependent DNA polymerase. Reverse transcriptase is an enzyme
    capable of synthesizing a strand of DNA (the first strand)
    complementary to the mRNA template (cDNA).

     The cDNA produced in this reaction can be used in cloning procedures,


    or assays for measuring gene expression levels such as PCR or Q-PCR.
    Components of cDNA synthesis

     For cDNA synthesis we need:

     RNA whithout gDNA


     Reverse Transcriptase enzyme
     primers (Random Hexamer Primer or Random Hexamer Primer)
     Ribolock RNase Inhibitor
     5X Reaction Buffer
     dNTP Mix
     Water, nuclease-free
    Reverse Transcriptase enzyme

     A reverse transcriptase (RT) is an enzyme used to generate complementary DNA


    (cDNA) from an RNA template, a process termed reverse transcription. This
    enzyme requires a primer, and for the generation of cDNAs from a collection of
    polyadenylated RNAs, we often use oligo(dT); a short stretch of
    deoxythymidylate residues.
     It is mainly associated with retroviruses. However, non-retroviruses also use RT
    (for example, the hepatitis B virus, a member of the Hepadnaviridae, which are
    dsDNA-RT viruses, while retroviruses are ssRNA viruses).
    Random Hexamer Primer

     Random Hexamer Primers consist of a mixture of oligonucleotides representing


    all possible hexamer sequences.
     Random Hexamer Primers are commonly used for priming single-stranded DNA
    or RNA for extension by DNA polymerases or reverse transcriptases.
     During cDNA generation, random priming gives random coverage to all regions
    of the RNA to generate a cDNA pool containing various lengths of cDNA.
     Random priming is incapable of distinguishing between mRNA and other RNA
    species present in the reaction.
     Primer Sequence:
    5´ – d (NNNNNN) –3´ N = G, A, T or C
    Random Hexamer Primer
    Oligo (dT) Primer

     Oligo (dT) Primer is suitable for use as a primer for first strand cDNA synthesis
    with a reverse transcriptase. The primer hybridizes to the poly-adenylated tail
    found on the 3´ end of most eukaryotic mRNAs. Oligo (dT)18 ensures that the 3´
    end of mRNAs are represented.
     Primer sequence: 5´-d (TTT TTT TTT TTT TTT TTT)-3´
     Oligo(dT) primers are a favorite choice for cDNA synthesis reactions because of
    their specificity for mRNA and because they allow many different targets to be
    studied from the same Cdna pool.
     A mixture of random hexamer primers and oligo(dT) may improve the sensitivity
    of cDNA synthesis.
    Oligo (dT) Primer
    RT PCR-types

    Two step RT PCR


    Traditionally, RT-PCR involves two steps: the RT reaction and PCR
    amplification.
    • RNA is first reverse transcribed in to complementary DNA (cDNA)
    Using an enzyme, reverse transcriptase.
    • The resulting cDNA is used as templates for subsequent PCR
    amplification using primers specific for one or more genes.
    One step RT-PCR
    • RT-PCR can also be carried out as one-step RT-PCR in which all reaction
    components are mixed in one tube prior to initiation of the reaction.
    • Although one-step RT-PCR offers simplicity and convenience and
    minimizes the possibility for contamination, the resulting cDNA cannot
    be used for detecting multiple messages from a single RNA sample as in
    two-step RT-PCR.
    One -step RT-PCR

    • One-step RT-PCR take mRNA targets (Up to 6 kb) and subjects them to
    reverse transcription and then PCR amplification in a single test tube.
    • 1.Select a one-step RT-PCR kit, which should include a mix with
    reverse transcriptase and the PCR system such as Taq DNA
    Polymerase and a proofreading polymerase.
    • 2.Obtain all necessary materials, equipment and instruments (kits should
    include a detailed list of necessary items).
    • 3.Prepare a reaction mix, which will include dNTPs, primers, template
    RNA, necessary enzymes and a buffer solution.
    • 4.Add the mix to a PCR tube for each reaction. Then add the template
    RNA.
    • 5.Place PCR tubes in the thermal cycler to begin cycling. The first cycle
    is reverse transcription to synthesize cDNA. The second cycle is initial
    denaturation. During this cycle reverse transcriptase is inactivated. The
    next 40 to 50 cycles are the amplification program, which consists of
    three steps: (1) denaturation, (2) annealing, (3) elongation.
    • 6.The RT-PCR products can then be analyzed with gel electrophoresis.
    Two-step RT-PCR

    • Two step RT-PCR, as the name implies, occurs in two steps. First the reverse
    transcription and then the PCR. This method is more sensitive than the one-step
    method. Kits are also useful for two-step RT-PCR. Just as for one-step, use only
    intact, high quality RNA for the best results. The primer for two-step does not
    have to be sequence specific.
    • Step one
    • 1.Combine template RNA,primer, dNTP mix, and nuclease-free water in a PCR
    tube.
    • 2.Add RNase inhibitor and reverse transcriptase to the PCR tube.
    • 3.Place PCR tube in thermal cycler for one cycle that includes annealing,
    extending and then inactivating reverse transcriptase.
    • 4.Proceed directly to PCR or store on ice until PCR can be performed.
    • Step two
    • 1.Add a master mix (containing buffer, dNTP mix, MgCl2, Taq polymerase and
    nuclease-free water) to each PCR tube.
    • 2.Add appropriate primer.
    • 3.Place PCR tubes in thermal cycler for 30 cycles of the amplification program,
    which include three steps: (1) denaturation, (2) annealing, (3) elongation.
    • 4.The RT-PCR products can then be analyzed with gel electrophoresis.
    Two-step RT-PCR
    Comparison of one-step & two-step RT-PCR procedures

    Comparison of one-step & two-step RT-PCR procedures

    Two-Step Procedure One-Step procedure

    • Oligo (dT) primer • Gene-specific primers


    Prime first-strand cDNA • Random hexamers
    with: • Gene-specific primers

    Flexibility Convenience
    • Choice of primer • Amplification enzymes premixed
    • Choice of amplification system with reverse transcriptase
    • Ability to save some RNA sample for later • Fewer pipetting steps and
    Provides use reduced changes of
    • Ability to optimize for difficult RT-PCR contamination.
    (combine with Platinum enzymes for
    higher specificity or combine with
    Platinum Pfx for greater fidelity)
    Recommended uses: • Ideal for detection or quantifying several • Ideal for analysis of large
    messages from, a single Sample numbers of samples
    • Ideal for real-time quantitative
    Program of TAKARA kit

    5X PrimeScript TM Buffer 4 μl

    PrimeScript TM RT Enzyme 1 μg
    Mix Incubated at
    Oligo dT Primer (50 μM) 1 μl 42°C for 25 min
    85°C for 7 sec
    Random 6 mers (100 μM ) 1 μl HOLD 4°C

    Total RNA 2-5 μg

    RNase Free dH2O ?


    Total 20 μl

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