SuperScript™ III First-Strand Synthesis System - FAQs

I am interested in generating cDNA from total RNA. What is the difference between SuperScript III Reverse Transcriptase and SuperScript III First Strand Synthesis System for RT-PCR?

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.

How long can I store the cDNA from my reverse transcription step?

You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.

How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

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.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. 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.

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 degrees 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 degrees 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.

How much RNA should be employed for first-strand cDNA synthesis?

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 µg total RNA is used in a typical 20-µL RT reaction.

Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.

Should I treat the cDNA with RNase H prior to downstream processing?

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 degrees 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.

What percentage of RNA is converted to cDNA when performing reverse transcription?

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.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

I'm setting up my RT reaction and am trying to decide whether I should use random primers, oligo(dT) primer, gene-specific primer, or oligo(dT)/random mix primers. What would you suggest?

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.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

The DTT in my reverse transcription kit has precipitated—can I still use it?

No, the DTT will need to be replaced.

Are SuperScript II and III RTs RNase H minus?

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.

Can I purchase the SuperScript III buffer separately?

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

Will adding EDTA prior to heat-inactivation of DNase I inhibit reverse transcription with SuperScript RT?

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.

In comparing the different SuperScript III kit formats, I notice that some utilize a 10X buffer and others a 5X. The recipes are also slightly different - why is this?

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.

Does SuperScript III exhibit TdT activity?

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

What is the difference between SuperScript III RT and the RT in the SuperScript VILO kit?

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

The SuperScript VILO cDNA Synthesis Kit (Cat. No. 11754050) 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 degrees C due to the helper proteins.

How much of the first-strand cDNA reaction should I load for PCR?

While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we'd recommend starting with 10% of the first-strand reaction for your PCR reaction.

SuperScript II RT is supplied as a 200 U/µL stock. We need to use 50 U for cDNA synthesis. Can we dilute the enzyme with 5X RT buffer to make it 50 units/µL?

You can dilute SuperScript II RT in 1X first-strand buffer if you plan to use it immediately. The enzyme is not stable in that buffer for extended periods. You can also dilute the enzyme in the storage buffer, which is 20 mM Tris HCl pH 7.5, 1 mM DTT, 0.01% NP40, 0.1 mM EDTA, 0.1 M NaCl 50% glycerol, and store it at -20 degrees C.

You may also consider using SuperScript III enzyme. This enzyme does not need to be diluted in order to perform optimally with a smaller amount of starting template.

What factors are important when designing degenerate primers?

Degenerate primers are a mixture of oligonucleotides that are all the same length but have more than one possible base at some of the positions. Degeneracy of the genetic code for the selected amino acid is generally the reason for needing/using degenerate primers for PCR. Degenerate primers are used in the following research scenarios:

(1) Locating the corresponding gene from the sequence of a protein that has been purified/isolated.

(2) Finding gene homologues across species. If the sequence of a particular gene is published across several species, there will be some universal homology and some degeneracy, so finding a homologue in a new species may require degenerate sequences for amplification.

(3) Locating related genes across species.

(4) Phylogenetic and evolutionary studies.

(5) Studying gene families.

Guidelines for designing degenerate primers
-Try to use primers that are a minimum of 20 bases.
-Selection of amino acids with minimal degeneracy is desired (amino acids requiring less than 4 or 6 codons). Serine, arginine, and leucine are the amino acids with the most wobbles.
-Consider codon bias for translation depending on the gene, organism, or virus!!!
-In addition to using degeneracy to compensate for the variance in the genetic code, it can also be used to substitute similar types of amino acids such as Glu--Asp and Arg--Lys.
-Degeneracy greater than 516-fold is not recommended. PCR has been performed using primers with higher degeneracy, but it is very inefficient. The degree of degeneracy depends on the number of degenerate positions and the number of bases used at these particular positions. E.g., ACC TGG CAA TTT CGG IGG CGT. Degree of degeneracy is 3 X 2 X 4 (3 possible bases at position 4, 2 possible bases at position 7, and 4 possible bases at position 16).
-Try to avoid degeneracy at the 3' end of the primer if possible. 6-9 base 5' extensions can be added. While not complementary to the template, these 5' extensions become incorporated into the amplified product at the second and all subsequent cycles. It increases the overall efficiency of PCR by increasing the stability of the duplex.
-Inosine-containing primers are also used because of the ability of this base to pair with all four bases. The drawback is that inosine (I) residues decrease the Tm of the primer.
-Avoid using degenerate primers to amplify large amplicons (>1,000 bp).
-Empirical testing of the designed primers is required. Consider a two step PCR when using degenerate primers, where the first 4 or so cycles are performed at a low annealing temperature and then linking it to a new program that steps up the annealing temperature 5-10 degrees C. Sometimes additional cycles are needed to generate sufficient product when performing degenerate PCR.

References
(1) Degenerate Primers in PCR, in PCR Protocols. A Guide to Methods and Applications. MA Innes, DH Gelfand, and JJ Sninsky eds (1990), chapter 5.
(2) PCR Protocols, Current Methods and Applications, in Methods in Molecular Biology. BA White ed (1993), chapter 31.

What is the highest temperature that SuperScript III , SuperScript II, MMLV, or ThermoScript can be used?

The optimal temperature for SuperScript III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. Optimal temperature for MMLV is 42 degrees C. ThermoScript RT shows optimal activity at 60 degrees C, and can be used at temperatures as high as 70 degrees 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 degrees 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)20 for cDNA synthesis when using an oligo (dT) primer for first-strand synthesis with these enzymes.