实验原理

The system consists of two major components: SuperScript™ III RT/ Platinum® Taq High Fidelity Enzyme Mix and 2X Reaction Mix. SuperScript™ III Reverse Transcriptase is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability.  The enzyme can synthesize cDNA at a temperature range of 45–60°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript™ III RT is not significantly inhibited by ribosomal and transfer RNA, it can be used to synthesize cDNA from total RNA.

Platinum® Taq  DNA Polymerase High Fidelity is an enzyme mixture composed of recombinant Taq  DNA polymerase, Pyrococcus species GB-D polymerase, and Platinum® Taq  antibodies, which block polymerase activity at ambient temperatures. The antibodies are denatured and polymerase activity is restored during the denaturation step in PCR cycling at 94° C, providing an automatic “hot start” in PCR and increasing sensitivity, specificity, and yield. Pyrococcus species GB-D polymerase is a proofreading enzyme that possesses a 3’ to 5’ exonuclease activity. Mixture of this enzyme with Taq  DNA polymerase results in a six-fold increase in fidelity over Taq  DNA polymerase alone and allows amplification of simple and complex DNA templates over a large range of target sizes.

The 2X Reaction Mix included in the kit consists of a proprietary buffer system that has been optimized for reverse transcription and PCR, and includes Mg2 , deoxyribonucleotide triphosphates (dNTPs), and stabilizers. A tube of 5 mM MgSO4 is included in the kit for further optimization of the Mg2  concentration. Sufficient reagents are provided for 25 or 100 amplification reactions of 50 µl each.

Note:   This kit has been optimized for end-point RT-PCR. For quantitative real-time RT-PCR, use the SuperScript™ III Platinum® One-Step Quantitative RT-PCR System.

实验试剂

实验设备

GeneAmp®PCR System 9600 and 2400

DNA Engine® PTC-200

实验材料

RNA

实验步骤

1. Program the thermal cycler so that cDNA synthesis is followed immediately with PCR amplification automatically.

2. Add the following to a 0.2-ml, nuclease-free, thin-walled PCR tube on ice. For multiple reactions, you can prepare a master mix to minimize reagent loss and enable accurate pipetting.

3. Gently mix and make sure that all the components are at the bottom of the amplification tube. Centrifuge briefly if needed. Depending on the thermal cycler used, overlay with silicone oil if necessary.

4. Place the reaction in the preheated thermal cycler programmed as described above. Collect the data and analyze the results.

注意事项

1. RNA Quality

1)             This kit has been optimized for use with 1 pg to 1 µg of total RNA.

2)             High quality intact RNA is essential for successful full-length cDNA synthesis.

3)             For low copy-number genes or longer targets, use more starting material (>10 ng total RNA).

4)             RNA should be devoid of any RNase contamination and aseptic conditions should be maintained.

5)             We recommend the PureLink™ Micro-to-Midi Total RNA Purification System or TRIzol® Reagent for isolation of total RNA. See the Materials section for ordering information. Oligo(dT) selection for poly(A) RNA is typically not necessary, although it may improve the yield of specific cDNAs.

2. Primer Design

6)             We recommend using gene specific primers (GSPs). We do not recommend using oligo(dT) or random primers, as they can generate nonspecific products in the one-step procedure and the amount of RT-PCR product may be reduced.

7)             A final primer concentration of 0.2 µM for each primer is generally optimal. However, for best results, we recommend performing a primer titration of 0.15-0.5 µM.

8)             Design primers that anneal to the mRNA sequence in exons on both sides of an intron or exon/exon boundary, to allow differentiation between the amplified cDNA and potential contaminating genomic DNA.

9)             Primers should not be self-complementary or complementary to each other at the 3´ ends.

3. Magnesium and dNTP Concentration

10)         MgSO4 is included in the 2X Reaction Mix at a final concentration of 1.2 mM, which works well for most targets. If needed, the magnesium concentration can further be optimized (usually between 1.2–2 mM) with the 5-mM MgSO4 provided in the kit.

11)         dNTPs are included in the 2X Reaction Mix at a final concentration of 200 µM, which is optimal for most reactions.

4. Reaction Setup

12)         Program the thermal cycler before setting up the reaction. The thermal cycler should be preheated to 45–60° C, depending on the temperature selected for cDNA synthesis.

13)         For difficult or high GC-content templates, use a 55–60° C cDNA synthesis temperature.

14)         Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them to the preheated thermal cycler and immediately start the RT-PCR program.

15)         Efficient cDNA synthesis can be accomplished in a 15–30-minute incubation at 45–60° C. For small targets, an incubation time of 5 minutes may be sufficient.

16)         SuperScript™ III RT is inactivated, Platinum ®  Taq DNA Polymerase High Fidelity is reactivated, and the RNA/cDNA hybrid is denatured during the 2-minute incubation at 94° C.

17)         The annealing temperature should be 10° C below the melting temperature of the primers used.

18)         The extension time varies with the size of the amplicon (approximately 1 minute per 1 kb of amplicon).

19)         For all targets up to 10 kb, 1 µl of SuperScript™ III RT/ Platinum ®  Taq High Fidelity Enzyme Mix is sufficient.