Reverse Transcription and RACE Support - Getting Started

MultiScribe™ Reverse Transcriptase (Cat. No. 4311235) and MuLV Reverse Transcriptase (Cat. No. N8080018) are essentially the same enzyme, a recombinant Moloney Murine Leukemia Virus (MuLV) Reverse Transcriptase. The only difference is in their recommended usage. MultiScribe™ Reverse Transcriptase is recommended for use with quantitative nuclease assays, and MuLV is recommended for use in traditional RT-PCR assays.

Yes, we do offer the SuperScript® CellsDirect™ cDNA Synthesis Kit which can be used for 1 cell up to 10,000 cells. This kit can also be used for frozen LCM samples but not formalin fixed/paraffin embedded tissues. Alternatively, you could also use our Ambion® Cells-to-CT™ kits which provides RT-PCR amplification directly from cell lysates. These kits are compatible with cultured cells and LCM samples.

The SuperScript® VILO™ cDNA Synthesis Kit contains a mix of SuperScript® III RT and helper proteins which help to increase the efficiency of the reverse transcription reaction and thus improve yield. The RT in the SuperScript® VILO™ kit is active at 42°C due to the helper proteins.

M-MLV RT, SuperScript® II RT, and SuperScript® III RT are stable for up to 2 years when stored at -20°C in a non frost-free freezer. Enzymes may remain at 4°C for up to 48 hours without loss of activity. However, fresh DTT is required for activity.

The optimal temperature for SuperScript® III RT is 50°C, and it can be used up to 55°C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60°C. The optimal temperature for SuperScript® II RT is 42°C, and it can be used up to 50°C. Optimal temperature for M-MLV RT is 42°C. ThermoScript™ RT shows optimal activity at 60°C, and can be used at temperatures as high as 70°C (for amplicons expected to be 1 kb or less). For PCR products expected to be greater than 1 kb, a maximum first-strand synthesis temperature of 60–65°C is suggested. Be sure your first-strand primer anneals at the high temperature, especially when gene-specific primers are used for high-temperature stable reverse transcriptases. We recommend oligo(dT)₂₅ for cDNA synthesis when using an oligo(dT) primer for first-strand synthesis with these enzymes.

No, if TdT activity is required please use our SuperScript® II RT.

SuperScript® III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085) contains the stand-alone enzyme and a vial each of 5X first-strand buffer and 100 mM DTT.

SuperScript® III First Strand Synthesis System for RT-PCR is a complete kit that provides the SuperScript® III Reverse Transcriptase and all the other components required for synthesis of first-strand cDNA from total or poly(A)- RNA. It includes:

• Superscript® III Reverse Transcriptase
• Oligo (dT)20 Primer
• Random hexamers
• 10X RT buffer
• 25 mM MgCl2
• 0.1 M DTT
• 10 mM dNTP Mix
• RNAseOUT™ Recombinant Ribonuclease Inhibitor
• E. coli RNAse H
• DEPC-treated water
• Total HeLa RNA control
• Sense control primer
• Anti-sense control primer

Note: The kit does not include the PCR amplification enzyme.

It is recommended to use the buffer that comes supplied with the enzyme. The reasons for the slight differences are that the kits were developed at different times, possibly by different R&D groups.

No. After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg²⁺:EDTA. EDTA chelates Mg²⁺ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg²⁺. If the reverse transcription buffer does not contain MgCl₂, add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl₂.

It is possible to generate full-length cDNA from mRNA attached to Dynabeads® magnetic beads. We recommend a thermostable reverse transcription kit, so that difficult regions with GC-rich secondary structures are accommodated. However, it is not possible to start the reaction by heating the mRNA on the beads because that will elute the mRNA (A:T base pairs are the least thermostable).

We have used ThermoScript™ RT, in-house, with oligo(dT)₂₅ on the beads as primers. The cDNA synthesis was performed according to the manufacturer's instructions. When using a thermostable reverse transcriptase and the oligo(dT)₂₅ as primer for first-strand cDNA synthesis, an initial step of incubation at 50°C for 5 min is necessary before proceeding at the recommended elevated temperature. This is to start the cDNA synthesis beyond the A:T hybridization point so that the mRNA doesn't fall off the beads. The resulting cDNA is covalently attached to the bead surface, and the beads with the attached cDNA can be used as template in multiple hybridization reactions.

The purpose of this step (heating at 65°C for 5 min) is to open up secondary structures in the RNA. If you want to use the oligo(dT)₂₅ on the beads as primers for your cDNA synthesis and generate solid-phase cDNA, you should omit this step. Start with 50°C (otherwise the mRNA will fall off the beads) then proceed to the 65°C step.

The following components are available as stand-alone items:

• Superscript® III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085)
• Oligo (dT)20 Primer (Cat. No. 18418020)
• Random hexamers (Cat. No. 48190011)
• 10 mM dNTP Mix (Cat. Nos. 18427013, 18427088)
• RNAseOUT™ Recombinant Ribonuclease Inhibitor (Cat. No. 10777019)
• E. coli RNAse H (Cat. Nos. 18021014, 18021071)

Yes, we sell a M-MLV RT buffer (Cat. No. 18057018), which works with M-MLV RT, SuperScript® II RT, and SuperScript® III RT.

Yes, we do offer Random Primers as a stand-alone item: Cat. No. 48190011.

Yes, we do offer E. coli RNAse H as a stand-alone item: Cat. Nos. 18021014 and 18021071.

These enzymes contain the domains of RNase H, but they have been mutated. In RNase H activity detection assays, we are not able to detect any RNase H activity.

The following reagents will inhibit SuperScript® II RT activity by at least 50%.

• 34% glycerol
• 4 μg/mL heparin
• 0.0025% (w/v) SDS
• 5% (v/v) formamide
• 17.0% (v/v) DMSO
• 4 mg/mL glycogen
• 30 mM guanidine-HCl
• 15 mM guanidine isothiocyanate
• 1 mM EDTA
• 2.5 mM NaPPi
• 0.4 mM spermidine

While retaining all the performance benefits of SuperScript® III RT, SuperScript® IV RT has the following additional benefits:

We do not recommend using SuperScript® III RT/ SuperScript® II RT 5X first-strand buffer with Superscript® IV RT. For optimal performance of Superscript® IV RT, we recommend using it with the 5X RT buffer supplied in the kit.

SuperScript IV RT functions exceedingly better than all tested enzymes in the presence of TRIzol Reagent, ethanol, SoluLyse reagent, guanidinium salts, LiCl, ammonium acetate, hematin, and humic acid. SuperScript IV RT is also able to perform in the presence of formalin, paraffin, and isopropanol.

We have found that SuperScript IV RT was able to produce a 12.3 kb cDNA product while all other reverse transcriptases produced smears and smaller products using a gene-specific primer.

SuperScript IV RT sustains 100% activity up to 56.4 degrees C and 70% activity up to 65 degrees C. The enzyme's ability to function at higher temperatures enables the reverse transcription of RNA targets with strong secondary structure.

No, we do not recommend using SuperScript IV RT Reaction Buffer with the SuperScript II RT or SuperScript III RT enzymes. For optimal performance, we recommended using the matching reaction buffer for each enzyme.

SuperScript IV VILO Master Mix is an upgrade from SuperScript VILO Master Mix and cDNA Synthesis Kit. It is designed to be used for cDNA synthesis in the two-step RT-qPCR applications for gene expression analysis.

SuperScript IV VILO Master Mix uses the newly developed SuperScript IV Reverse Transcriptase (RT) in the optimized master mix formulation, whereas SuperScript VILO Master Mix and kits use SuperScript III Reverse Transcriptase. SuperScript IV RT has significantly improved performance profile over SuperScript III RT in sensitivity, yield, processivity and ability to synthesize cDNA efficiently from a wide variety of samples of suboptimal conditions. In SuperScript IV VILO Master Mix, the performance advantages of SuperScript IV RT enzyme are successfully retained in the master mix format to provide a product with the performance improvement in the most convenient format.

The recommended reaction temperature for SuperScript IV VILO Master Mix is 50 degrees C. R&D has tested the stability of SSIV VILO at higher temperatures and it works very well even at 65 degrees C.

SuperScript IV VILO Master Mix has increased thermostability, allowing the RT incubation temperature to be raised to deal with the structural complexity often associated with GC-rich templates. Our tests show that compared to other cDNA synthesis kits or master mixes, the raised reaction temperature leads to minimal loss of cDNA synthesis efficiency for SuperScript IV VILO Master Mix, suggesting that it is the best choice if high reaction temperature is needed for cDNA synthesis from GC-rich templates.

This is to help ensure that the biases from using either oligo (dT)18 or random hexamer primers alone will be eliminated, although the data indicate that using random hexamers alone provides quantitation just as good as the combination of the two primers. Some scientists want the combination because they feel it could be better for data quality.

For the 50 reaction SKUs, the no RT control can be for 50 reactions. For the 500 reaction SKUs, the no RT control can be for 250 reactions, which is way more than what is needed for control reaction purpose.

The ezDNase enzyme is highly specific for double-stranded DNA and does not cause damage to RNA and single stranded DNA. The high specificity of its action makes the deactivation step largely irrelevant in the subsequent RT reactions. ezDNase enzyme can be inactivated at 55 degrees C incubation for 2 mins if needed.

Include a control reaction where the RNA has not been incubated with reverse transcriptase to test for specificity. If this RNA gives a PCR product, it is most likely generated from genomic DNA contamination. Alternatively, a primer set spanning two different exons can be designed such that the PCR product from the cDNA would be of a different size compared to a product generated from genomic DNA. Primers may also be designed to span exon/exon junctions. These primers are not likely to amplify from genomic DNA templates. For DNase treatment of RNA, we recommend using DNase I, Amplification Grade (Cat. No. 18068015) or an equivalent product.

No, the DTT will need to be replaced.

Yes, we offer three different kits which may suit your needs:

1. SuperScript® Plasmid System for cDNA Synthesis and Cloning
   • Generating and cloning cDNA into a CMV-containing Gateway®-compatible vector (pCMVSport6) for mammalian screening
   • mRNA required as starting material (must have poly(A) tail)
   • Can probe screen in E. coli but not expression screen
   • Resulting dsDNA is unidirectional with Sal I / Not I sites
2. SuperScript® Choice System for cDNA Synthesis
   • Will work on any RNA source
   • Can use random, oligo(dT), or combination
   • Will result in cDNA with EcoRI ends which can be cloned into any vector (plasmid or phage) that has EcoRI sites
   • Good for generation of cDNA libraries
   • Resulting dsDNA will clone bidirectionally
3. SuperScript® Double-Stranded cDNA Synthesis Kit
   • For generating blunt-ended, double-stranded cDNA
   • Will work for total RNA or mRNA as starting material

One-step RT-PCR is convenient, and less prone to contamination as there is less opportunity for pipetting error. This method is also faster than two-step. However, the cDNA cannot be archived, and fewer genes can be analyzed. Two-step RT-PCR gives you the ability to archive cDNA, analyze multiple genes, and gives greater flexibility.

Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

This depends highly on the quality of the sample. mRNA itself makes up 1–5% of total RNA. Depending on the primer and enzyme used, reverse transcription can covert >70% of that into cDNA.

Some feel that the RNA in the RNA:DNA duplex after reverse transcription will inhibit PCR primers from annealing and amplifying the cDNA. The RNA is still present when using RNase H–mutant RTs. RNase H frees the cDNA from the RNA. On the other hand, some feel that the 95°C denaturing step will cause the RNA primers to fall off the DNA and therefore RNase H treatment is not necessary. Therefore, this step is optional. For cloning of larger fragments, Rnase H treatment can be beneficial.

The amount of RNA template for a cDNA synthesis is highly flexible and depends upon the amount of sample available and an individual’s need. In general, 1 microgram total RNA is used in a typical 20-μL RT reaction.

The volume will depend on the starting amount of RNA used for first-strand synthesis, and the abundance of the target gene. We recommend starting with 10% of the first-strand reaction in a 50-μL PCR reaction. More than 10% may inhibit downstream reactions.

RNAse-free DNase treatment of the RNA can reduce DNA to undetectable levels. We recommend using our DNase 1, Amplification Grade (Cat. No. 18068015).

We recommend using our Purelink® RNA Mini Kit (Cat. No. 12183025) or TRIzol® Reagent (Cat. No. 15596026) to isolate your RNA. Oligo(dT) selection for mRNA is typically not necessary, although it may improve the yield of specific cDNAs.

The inhibitor acts as a safeguard against degradation of target RNA due to ribonuclease contamination of the RNA preparation.

If amplification products are generated in the control tube/well that contains no reverse transcriptase (i.e., the no-RT control), it may be necessary to eliminate residual genomic DNA from the RNA sample. Use the following protocol to remove genomic DNA from the total RNA preparation.

Add the following to an autoclaved 0.5 mL microcentrifuge tube on ice:

1. Total RNA, ideally, less than or equal to 1 μg. (See Note 1 below.)
2. 1.0 μL of 10X DNase buffer (200 mM Tris, pH 8.3, 500 mM KCl, 20 mM MgCl₂).
3. 0.1 U–3.0 U of DNase I (RNase-free, Cat. No. 18047019) or 1.0 U Dnase I, Amplification Grade (Cat. No. 18068015. (See Note 2 below.)
4. Bring volume up to 10 μL with DEPC-treated water.
5. Incubate at room temperature for 15 min. (See Note 3 below.)
6. Terminate the reaction by adding 1 μL 25 mM EDTA and heat 10 min at 65°C. (See Note 4 below.)
7. Place on ice for 1 minute.
8. Collect by brief centrifugation. This mixture can be used directly for reverse transcription.

Please note the following:

1. To work with higher quantities of RNA, scale up the entire reaction linearly. Do not exceed 2 μg RNA in the 10 μL reaction. More RNA will increase the viscosity of the solution and prevent the DNAse I from diffusing and finding the DNA.
2. 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.
3. It is important not to exceed the 15 minute incubation time or the room temperature incubation. Higher temperatures and longer times could lead to Mg²⁺-dependent hydrolysis of the RNA.
4. This procedure requires careful pipetting of all solutions so that the concentration of divalent metal cation (Mg²⁺) is controlled.
5. Because the DNAse I must be heated to 65°C to inactivate the enzyme, the concentration of free divalent metal ions must be low enough (less than 1 mM) after addition of the EDTA to prevent chemical hydrolysis of the RNA. See references below.

After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg²⁺ :EDTA. EDTA chelates Mg²⁺ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg²⁺. If the reverse transcription buffer does not contain MgCl², add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl².

References on RNA hydrolysis:
Molekulyarnaya Biologiya (1987) 21:1235-1241.
References on the mechanism of hydrolysis by other cations:
Eichorn GL and Butzov JY (1965) Biopolymers 3:79.
Butzov JY and Eichorn GL (1965) Biopolymers 3:95.
Farkas WR (1968) Biochim Biophys Acta 155:401.
The authors of the first paper express the opinion that the mechanism of the nonspecific hydrolysis by cations which proceeds through 2′,3′ cyclic phosphate formation is similar to that of specific hydrolysis such as RNA splicing.

To check the RNA for integrity, analyze 500 ng of your RNA by agarose/ethidium bromide gel electrophoresis. You may use a regular 1% agarose gel or a denaturing agarose gel. For total RNA you should see the 28S and 18S rRNA bands. mRNA will appear as a smear from 0.5 to 12 kb. The 28S band should be twice the intensity of the 18S band. If you do not load enough RNA, the 28S band may appear to be diffuse. If you are using a denaturing gel, the rRNA bands should appear very clear and sharp. The 28S band should run at 4.5 kb and the 18S band should run at 1.9 kb.

If you are performing either 5′ or 3′ RACE, you will need one gene-specific primer and if you are performing both 5′ and 3′ RACE, you would need two gene-specific primers. The primers should follow the rules stated below:

• 50–70% GC content to obtain a high annealing temp (>72°C)
• 23–28 nucleotides in length to increase specificity of binding
• Low-GC content at 3′ ends to minimize extension by DNA polymerase at non-target sites (no more than two G or C residues in the last five bases)
• No self-complementary sequences within the primer or no sequence complementary to the primers supplied in the kit, especially at the 3′ end
• Annealing temperature greater than 72°C—using primers with a high annealing temperature will help to improve specificity of your PCR

The following formula will help you to approximate the annealing temperature of your primer:
4 x (G+C) + 2 x (A+T) = annealing temperature (approximate Tm) where G, C, A, or T represent the number of these bases in the primer sequence.

If the reaction gives a specific product and the control reaction does not, your band is probably real. The only way to be sure is to sequence the product.

Alternative splicing, an alternative polyadenylation site, and alternative start sites can yield legitimate multiple bands. Sequencing will help to resolve any uncertainty.

Here are our suggestions to optimize your RACE reaction:

• Use high-quality, intact RNA
• Use an RNA sample in which your gene is expressed at high levels
• Do not exceed the recommended amount of input material in each step
• Optimize annealing temperatures for PCR
• Include recommended controls

3′ RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for PCR. In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then amplified by PCR using a gene-specific primer (GSP) that anneals to a region of known exon sequences and an adapter primer that targets the poly(A) tail region. This permits the capture of unknown 3′-mRNA sequences that lie between the exon and the poly(A) tail.

The CIP enzyme will not recognize triphosphate nucleotides. If the ends of your product have anything other than a monophosphate, the CIP will not dephosphorylate it, and later in the protocol the RNA ligase will not bind the RNA oligo to this 5′ triphosphate. If you follow the protocol provided for obtaining full-length capped messages, you will not get any product at all from 5′ triphosphate-RNA material.

The Adapter Primer (AP), Universal Amplification Primer (UAP), and Abridged Universal Amplification Primer (AUAP) have been discontinued as stand-alone items. Their sequences can be found on Page 4 of the manual.

The 5’ RACE Abridged Anchor Primer (AAP), Universal Amplification Primer (UAP), and Abridged Universal Amplification Primer (AUAP) have been discontinued as stand-alone items. Their sequences can be found on Page 4 of the manual.

Nested PCR requires two separate amplifications—the first one using one set of PCR primers and the second one using internal "nested" primers plus 1% or less of the first PCR reaction as a template. Nested PCR is used when the target is present in low abundance or when nonspecific PCR products are being produced along with the specific product. Semi-nested PCR is used when there is only enough sequence information to make a primer internal to one end of the primary PCR product such as in RACE.