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  • ProtoScript® First Strand cDNA Synthesis Kit

    Description

    ProtoScript® First Strand cDNA Synthesis Kit features two optimized mixes, M-MuLV Enzyme Mix and M-MuLV Reaction Mix. M-MuLV Enzyme Mix combines M-MuLV Reverse Transcriptase and Murine RNase Inhibitor, while M-MuLV Reaction Mix contains dNTPs and an optimized buffer. The kit also contains two optimized primers for reverse transcription and nuclease-free water. An anchored oligo-dT primer [d(T)23VN] forces the primer to anneal to the beginning of the polyA tail. The optimized Random Primer Mix provides random and consistent priming sites covering the entire RNA template including both mRNAs and non-polyadenylated RNAs. The first strand cDNA product generated is more than 10 kb (Figure 1).

    General Information for Successful cDNA Synthesis:

    Template RNA
    Intact RNA of high purity is essential for sensitive RT-PCR detection. RNA should have a minimum A260/A280 ratio of 1.7 or higher. 

    Either total RNA or mRNA can be used in the reverse transcription reaction. Total RNA is generally sufficient for most RT-PCR analyses. However, if desired mRNA can be easily obtained using a PolyA Spin mRNA Isolation Kit (NEB #S1560) or Magnetic mRNA Isolation Kit (NEB #S1550).

    The amount of RNA required for detection depends on the abundance of the transcript of interest. In general 1 ng to 1 μg total RNA or 0.1-100 ng mRNA are recommended.

    First Strand cDNA Synthesis Reaction
    Denaturation of RNA and primer at 70°C for 5 minutes can remove secondary structures that may impede long cDNA synthesis. However, this step can be omitted in some cases (unpublished results). 

    We recommend incubation at 42°C for one hour for maximum yield and length. However, many targets can be detected after a much shorter incubation time. For example, 5 minutes incubation is enough for a 2 kb cDNA synthesis.

    Choice of Primers for Reverse Transcription
    Oligo d(T) priming is preferred for most applications because it ensures that all cDNA copies terminate at the 3´ end of the mRNA and produces the longest contiguous cDNA. An anchored oligo-d(T) primer [d(T)23VN] forces the primer to anneal to the start of the polyA tail, thereby preventing priming at internal sites in the polyA tail (1). However, two other priming choices are possible if desired.

    The Random Primer Mix is an optimized mix of hexamer and d(T)23VN primers. It provides random priming sites covering the entire RNA templates including both mRNAs and non-polyadenylated RNAs (such as ribosomal RNAs). The Random Primer Mix yields shorter cDNAs on average and can be used for the detection of multiple short RT-PCR products. Random Primer Mix offers good performance in a wide range of RNA templates. 

    When a gene-specific primer is used in a cDNA synthesis reaction, the cDNA product can be used only for amplification of that transcript. This priming method gives good results when the amount of RNA is limiting (below 10 ng) and only one particular cDNA is desired.

    Recommended primer concentration:
    PRIMER -- Final conc.
    OLIGO d(T)23VN -- -- 5 μM
    RANDOM PRIMER MIX -- 6 μM
    SPECIFIC PRIMER -- 0.1–1 μM





    Figure 1:
    First strand cDNA synthesis was carried out with 1X M-MuLV Enzyme Mix at 42°C using 2 μg of human spleen total RNA. Negative control reactions were carried out with 1X M-MuLV Reaction Mix. A fraction of the first strand cDNA product was used to amplify sequences specific for three different messenger RNAs using 1X LongAmp™ Taq 2X Master Mix (NEB #M0287 ). Lane 1: 1.1 kb of beta-actin gene. Lane 2: noRT control of 1.1 kb of beta-actin gene. Lane 3: 4.7 kb of Xrn-1 gene. Lane 4: noRT control of 4.7 kb of Xrn-1 gene. Lane 5: 9.8 kb of guanine nucleotide exchange factor p532. Lane 6: noRT control of 9.8 kb of guanine nucleotide exchange factor p532. Marker M is 2-Log DNA Ladder (NEB #N3200 ).

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Oligo d(T)23 VN-2050 μM
    Nuclease-free Water
    M-MuLV Enzyme Mix10X
    M-MuLV Reaction Mix2X
    Random Primer Mix-2060 μM

    Properties and Usage

    Storage Temperature

    -20°C

    Quality Control

    Quality Assurance Statement

    • The performance of ProtoScript M-MuLV First Strand cDNA Synthesis Kit is tested in an RT reaction using human Jurkat total RNA with primer d(T)23VN. The sensitivity of the kit is verified by the detection of GAPDH transcript in 20 pg total RNA after 35 cycles. The length of cDNA achieved is verified by the detection of a 5.5 kb amplicon of the p532 gene.

    References

    1. Liao, J. and Greg, Z. (1997). Biotechniques. 23, 368-370.
    2. Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). (pp. 8.46-8.53 and 11.37-11.42). Cold Spring Harbor.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].
    1. Why am I getting a low yield of cDNA?
    1. First Strand cDNA Synthesis Protocols (E6300)

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    Usage Guidelines & Tips