SuperScript III First-Strand Synthesis SuperMix

Kit Contents

The components of the SuperScript First-Strand Synthesis System for RT-PCR are listed below. Components are provided in sufficient quantities to perform a total of 50 separate reactions; each converts from 1 ng up to 5 µg of total RNA into first-strand cDNA.
 
The SuperScript First-Strand Synthesis System for RT-PCR is stored at –20°C.

Quality Control

A minimum of 40 ng of a 1181-bp RT-PCR product was obtained from 10 pg of total HeLa RNA and human GAPDH primers. A minimum of 200 ng of a 5.6-kb RT-PCR product was obtained from 100 ng of total HeLa RNA and human DNCH primers

The following procedure is designed to convert 0.1 pg to 5 μg of total RNA or 0.1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA:

1. Mix and briefly centrifuge each component before use. Preheat the thermal cycler to 65°C.


2. Combine the following in a 0.2-ml thin-walled PCR tube on ice:
   
   

Component	Amount
up to 5 μg total RNA	n μl
Primer (50 μM oligo(dT)20, or 2 μM gene-specific primer, or 50 ng/μl random hexamers)	1 μl
Annealing Buffer	1 μl
RNase/DNase-free water	to 8 μl




3. Incubate in a thermal cycler at 65°C for 5 minutes, and then immediately place on ice for at least 1 minute. Collect the contents of the tube by brief centrifugation.


4. Add the following to the tube on ice: 2X First-Strand Reaction Mix 10 μl SuperScript III/RNaseOUT Enzyme Mix 2 μl


5. Vortex the sample briefly to mix, and collect by brief centrifugation. Incubate as follows: Oligo(dT)20 or GSP primed: 50 minutes at 50°C Random hexamer primed: 5–10 minutes at 25°C, followed by 50 minutes at 50°C


6. Terminate the reactions at 85°C for 5 minutes. Chill on ice.

Store the cDNA synthesis reaction at -20°C, or proceed directly to PCR.

Required Materials

• 0.2-ml, nuclease-free thin-walled PCR tubes
• Thermal cycler, preheated to 65°C
• Ice
• RNase/DNase-free water
• Microcentrifuge
• Vortex mixer

RNA

• High-quality, intact RNA is essential for full-length, highquality cDNA synthesis. This kit is designed for use with 0.1 pg to 5 μg of total RNA or 0.1 pg to 500 ng of poly(A)+ RNA.
• RNaseOUT Recombinant RNase Inhibitor is included in the Enzyme Mix to safeguard against degradation of target RNA due to ribonuclease contamination of the RNA preparation.
• To isolate total RNA, we recommend Invitrogen Ambion TRIzol Reagent (Cat. Nos. 15596-026/-018) or the Micro-to-Midi Total RNA Purification System (Cat. no. 12183-018). Isolation of poly(A)+ RNA is typically not necessary, although it may improve the yield of specific cDNAs; we recommend the Invitrogen FastTrack 2.0 mRNA Isolation Kit (Cat. no K1593-02).
• Small amounts of genomic DNA in the RNA preparation may be amplified along with the target cDNA. If your application requires removal of all genomic DNA from your RNA preparation, we recommend using DNase I, Amplification Grade (Catalog no. 18068-015). DNase I, Amplification Grade, has been extensively purified to remove trace ribonuclease activities commonly associated with other “RNase-free” enzyme preparations, and does not require the addition of placental RNase inhibitor.

cDNA Synthesis Reaction Conditions

• For difficult or high GC-content templates, use a 55°C cDNA synthesis temperature.
• Keep all components, reaction mixes, and samples on ice. After preparation of the annealing mixture, transfer the mixture to the preheated thermal cycler.

Primers

First-strand cDNA synthesis can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs):

• Random hexamers are the most nonspecific priming method, and are typically used for difficult or high GC-content mRNA. Using random hexamers, all RNAs in a population are templates for first-strand cDNA synthesis, and PCR primers confer specificity during PCR.

• Note:  For most RT-PCR applications, 50 ng of random hexamers per 5 μg of total RNA is adequate. Increasing hexamers to 250 ng per 5 μg of RNA may increase yield of small PCR products (<500 bp), but may decrease the yield of longer PCR products and full-length transcripts.

• Oligo(dT), a more specific priming method, is used to hybridize to 3′ poly(A) tails, which are found in the vast majority of eukaryotic mRNAs. Since poly(A)+ RNA constitutes approximately 1% to 2% of total RNA, the amount and complexity of cDNA is considerably less than with random hexamers. We recommend using the oligo(dT)20 provided in the kit.

• Note:  Oligo(dT) is recommended over random hexamers or GSPs for new mRNA targets. Oligo(dT) produces an RT-PCR product more consistently than random hexamers or GSPs.

• The most specific priming method uses a gene-specific primer (GSP) for the sequence of interest. First-strand synthesis can be primed with the PCR primer that hybridizes nearest to the 3´ terminus of the mRNA. Note that some GSPs fail to prime cDNA synthesis even though they work in PCR on DNA templates. If gene-specific priming fails in RT-PCR, repeat first-strand synthesis using oligo(dT) as the primer.

Guidelines for PCR

The first-strand cDNA from the synthesis reaction may be amplified directly using PCR. We recommend using 10% of the first-strand reaction (2 μl) for PCR. However, for some targets, increasing the amount of first-strand reaction to up to 10 μl may result in increased product yield.

We recommend the following DNA polymerases:

• Invitrogen Platinum Taq DNA Polymerase provides automatic hot-start conditions for increased specificity and sensitivity. It is recommended for targets up to 4 kb.
• Invitrogen Platinum Taq DNA Polymerase High Fidelity provides increased fidelity and higher yields for targets up to 15 kb.
• Invitrogen Platinum Pfx DNA Polymerase possesses a proofreading 3´ to 5´ exonuclease activity and provides maximum fidelity for PCR. It is recommended for targets up to 12 kb.