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ProtoScript® II RT-PCR Kit |
 | | | | ProtoScript® II RT-PCR Kit |
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Prices are in US dollars and valid only for US orders.
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- 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
Description:
The ProtoScript II 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. It the first step, M-MuLV Reverse Transcriptase (RT) is used to extend a random nonamer 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 a ready-to-use Taq 2X Master Mix, which is robust and best suited for detecting the presence or absence of a transcript (1).
Method Overview
There are several important factors to consider when setting up 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. The oligo dN9 provides random priming sites throughout the entire RNA for unbiased representation of all regions including both mRNAs and non-polyadenylated RNAs (such as ribosomal RNAs). It yields shorter cDNAs on average and can be used for the detection of multiple short RT-PCR products. Random priming is more sensitive to the ratio of primer to RNA concentration, especially when low amounts of RNA template are used.
OR
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.
The recommended primer amount for a 20 µl cDNA synthesis reaction is as follows:
dT23VN Primer (50 µM) - 100 pmol (2 µl)
oligo dN9* (15 µM) - 30 pmol (2 µl)
Specific Primer - 10 - 20 pmol
* Recommended primer amount is for 10 ng to 1 µg total RNA. Outside this range, the amount of dN9 needs to be adjusted for optimal yields of cDNA.
cDNA Synthesis Reaction:
The 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).
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.
If cloning the RT-PCR product is desired, we recommend the USER Friendly Cloning Kit (NEB #E5500). In this case, one primer for PCR must begin with the sequence 5'-GGAGACAU followed by the gene-specific sequence; the other primer for PCR must begin with the sequence 5'-GGGAAAGU followed by the gene-specific sequence. After treatment with the USER™ Enzyme, the PCR product is annealed to the supplied linearized plasmid vector, pNEB206A. As little as 0.8 ng/µl PCR product can be directly used in a 30 minute reaction and is ready for transformation.
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 and 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 may 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.
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).
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. Reactions were set up with and without M-MuLV Reverse Transcriptase (-RT Control). After 30 cycles of amplification using 1/20th of the cDNA product, 5 µl was analyzed on a 1% agarose gel. 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.
Kit Components:
Taq 2X Master Mix
Control (GAPDH) Primer Set
Control Total RNA (rat liver)
Deoxynucleotide Solution Mix
Detailed Instruction Manual
M-MuLV Reverse Transcriptase
M-MuLV Reverse Transcriptase Reaction Buffer
Oligo d(T)23 VN
Random Primer 9
RNase Inhibitor
Storage Conditions
Storage Temperature:
-20°C
FAQs
- What are the applications for the ProtoScript® II RT-PCR Kit?
- Can I use the ProtoScript® II RT-PCR Kit to detect long messenger RNAs?
- What is the difference between the ProtoScript® RT Kit and the ProtoScript® II RT-PCR Kit?
- What are critical factors for good RT reactions with the ProtoScript® II RT-PCR Kit?
- What are the critical factors for PCR reactions when using the ProtoScript® II RT-PCR Kit?
- Can I use 2-step PCR protocols with the ProtoScript® II RT-PCR Kit?
References
- Khan S. A. et al. (1991) Nucleic Acids Res., 19, 1715.
- Liao, J. and Gong, Z. (1997) Biotechniques, 23, 368-370.
Reagents Sold Separately
Taq 2X Master Mix
Deoxynucleotide Solution Mix
M-MuLV Reverse Transcriptase
M-MuLV Reverse Transcriptase Reaction Buffer
Oligo d(T)23 VN
Random Primer 9
RNase Inhibitor
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