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?

We recommend using ezDNase (Cat. No. 11766051). ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55 degrees C). These features make ezDNase Enzyme an excellent choice for genomic DNA removal prior to reverse transcription reactions.

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?

RNase H treatment is not always necessary. Many PCR reactions work without it. However, for cDNA synthesized with RNase H-deficient reverse transcriptases (like SuperScript II, III, and IV), RNA/cDNA hybrids—especially GC-rich ones—may not denature well, reducing PCR sensitivity. RNase H treatment can help in such cases. Additionally, RNase H treatment is beneficial for cloning larger fragments.

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 for reduced RNase H activity. 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

EDTA chelates Mg2+ molecules on a 1:1 molar basis. If the amount of EDTA used for DNase I inactivation does not exceed the amount of Mg2+ present in the DNase reaction buffer, the resulting RNA solution can be used directly in the RT reaction. Otherwise, the sample should be purified to remove excess EDTA. Alternatively, consider using DNase removal methods that do not rely on EDTA or heat inactivation to avoid interference. To reduce the risk of compromised cDNA synthesis, we recommend performing gDNA removal with ezDNase Enzyme (Cat. No. 11766051) which is specific to dsDNA and heat-labile, hence does not require harsh inactivation conditions.

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?

SuperScript III exhibits low TdT activity. If TdT activity is required, use our SuperScript IV RT or SuperScript IV Template Switching RT Master Mix.

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 units/µL stock. We need to use 50 units 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 (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.

Also consider using SuperScript IV reverse transcriptase. 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 MMLV, SuperScript II, SuperScript III, or SuperScript IV RTs can be used?

The optimal temperature for for MMLV is 42 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. 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 IV RT is 50 degrees C, but can be used up to 65 degrees C.