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Product Information
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 Inhibitor,
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:
Template RNA
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.
RNA-priming Choices
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:
- RNA without poly(A) tail
- RNA with strong secondary structures
- Partially degraded RNA samples
- Targets regions at 5´ end of a long messenger RNA transcript
2. 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 amount for a 20 μl cDNA synthesis reaction:
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 may be omitted in many cases (Xu, Y., unpublished observations).
PRIMER -- Amount
Oligo d(T)23VN PRIMER (50 μM) -- 100 pmol (2 μl)
RANDOM PRIMER MIX (60 μM) -- 120 pmol (2 μl)
SPECIFIC PRIMER -- 10–20 pmol
We recommend incubation at 42°C for one hour for maximum cDNA yield and length. However, many targets can be detected after a much shorter incubation time. For example, a 10 minute incubation time is enough for a 2 kb cDNA synthesis.
PCR Primers
For best results, specific primers for PCR should be designed with the aid of a primer design computer program, such as PrimerSelect™ (DNAStar Inc, Madison, MI) or Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www. cgi). To minimize complications introduced by contaminating genomic DNA, use primers that span an exon-exon boundary of the mRNA. In general, primers with 40–60% GC and temp. of 55–68°C are preferred.
PCR Amplification- Most targets can be efficiently amplified using 1/10th (2 μl of 20 μl) of the cDNA synthesis reaction, or less of the cDNA product (2).
- A final concentration of 0.2 μM for each primer is recommended for PCR; however, it can vary between 0.05 μM and 1 μM.
- The recommended extension step using the Taq 2X Master Mix is 68°C with an extension time of one minute per kb.
- The final Mg2+ concentration of the Taq 2X Master Mix is 1.5 mM, which is optimal for most RT-PCR applications. However, the Mg2+ concentration can be further optimized in 0.2 mM increments.
- A manual hot-start my increase PCR sensitivity and yield. This is done by assembling reactions in thin wall 0.2 ml PCR tubes placed on ice. Tubes are then transferred to a PCR machine with a block preheated at 95°C, and cycler is immediately started.
Figure 1:
RT-PCR amplification of the GAPDH gene from human spleen total RNA. First strand cDNA was carried out in the presence of oligo dT23VN, and about 1/10th of the cDNA reaction was amplified in a 35-cycle PCR reaction using the Taq 2X Master Mix (-RT control not shown).
Figure 2:
Amplification of different regions of human guanine nucleotide exchange factor p532 (15,164 bp, GenBank accession number U50078). Approximately 2 μg of human spleen total RNA was reverse transcribed using dT23VN. After 30 cycles of amplification using 1/20th of the cDNA product, 5 μl was analyzed on a 1% agarose gel. (Note: In the -RT control reactions, no reverse transcriptase was added.) Lane 1: 2-log DNA Ladder Lane 2: -RT Control of 1.2 kb fragment Lane 3: 1.2 kb fragment approx. 15 kb from 3´ end Lane 4: -RT Control of 0.9 kb fragment Lane 5: 0.9 kb fragment approx. 1.1 kb from 3´ end
Figure 3:
Analysis of control reactions on a 1% agarose gel. Lane 1: 2-log DNA Ladder, Lane 2: 5 μl of the -RT control reaction, Lane 3: 5 μl of the positive control reaction.Highlights
Sensitivity — detect transcripts as low as 20 pg total RNA
Robustness — reverse transcription of long cDNA products (up to 15 kb)
Flexibility — detection of multiple targets from 1 reaction
Robust PCR — includes Taq 2X Master Mix with optimal amplification features- Product Categories:
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