The Protoscript® Taq RT-PCR Kit is designed for the
sensitive detection of mRNAs in a two-step process. Each reaction is optimized
for maximum results, leading to greater sensitivity and higher yield. Multiple
transcripts can be detected from a single first‑strand cDNA synthesis.
Semi-quantitative analysis of the mRNA level can be achieved by agarose gel
electrophoresis. In the first step, M-MuLV Reverse Transcriptase (RT) is used to
extend a random primer, anchored oligo-dT primer, or gene-specific primer
annealed to an RNA sample. In the second step, PCR amplification is performed in
a separate tube using gene-specific primers. This Kit includes Murine RNase
which provides better protection of RNA against RNAses than human RNAse Inhibitor. A Random Primer Mix is introduced to provide even and consistent coverage of the RNA template population across a wide range of RNA template concentration. A ready-to-use Taq 2X Master Mix is provided for its convenient and consistent amplification performance.
Important Factors for Successful RT-PCR Reactions:
Intact RNA of high purity is essential for sensitive RT-PCR detection.
Both total RNA and mRNA can be used in the reverse transcription reaction. Total RNA is generally sufficient for most RT-PCR analysis. However, if desired, mRNA can be easily obtained using a PolyA Spin mRNA Isolation Kit (NEB #S1560).
The amount of RNA required for detection depends on the abundance of the transcript of interest. In general 10 ng to 1 μg of total RNA or 1 ng to 100 ng of mRNA are recommended.
Oligo-dT priming is recommended for most applications. It ensures that all cDNA copies terminate at the 3´ end of the mRNA and produces the longest contiguous cDNA. An anchored oligo-dT primer (dT23VN) 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.
1. Random primers provide random priming sites throughout the entire RNA templates including both mRNAs and non-polyadenylated RNAs such as ribosomal RNAs. Traditional random priming by hexamer is sensitive to the ratio of primer to RNA amount. In contrast, Random Primer Mix is an optimized mixture of hexamers and anchored-dT primer (dT23VN). A mixture of hexamers and anchored-dT primer provides even and consistent coverage of the RNA template population across a wide range of RNA template concentration. We recommend using Random Primer Mix for reverse transcription of the following RNA templates:
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|M-MuLV Reverse Transcriptase Reaction Buffer||-20||10 X|
|Taq 2X Master Mix||-20||2 X|
|M-MuLV Reverse Transcriptase||10,000 units/ml|
|RNase Inhibitor, Murine||-20||40,000 units/ml|
|Control Total RNA (rat liver)||500 µg/ml|
|Control (GAPDH) Primer Set||10 µM each|
|Oligo d(T)23 VN||-20||50 µM|
|Random Primer Mix||-20||60 µM|
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