Invitrogen™ SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR

Catalog No.	No. of Reactions
11-752-250	250 Reactions
11-752-050	50 Reactions

Catalog No. 11-752-250 Supplier Invitrogen™ Supplier No. 11752250

Description

SuperScript III First-Strand Synthesis SuperMix for qRT-PCR provides the high-temperature capability of SuperScript III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first-strand cDNA for use in real-time quantitative RT-PCR (qRT-PCR).

SuperScript III First-Strand Synthesis SuperMix for qRT-PCR provides the high-temperature capability of SuperScript III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first-strand cDNA for use in real-time quantitative RT-PCR (qRT-PCR). The simple, time-saving reaction set-up uses just two tubes: a 2X reaction mix and an enzyme mix.

Enzyme mix
SuperScript III Reverse Transcriptase, included in the RT enzyme mix, is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can be used to synthesize cDNA at a temperature range of 42–60°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. RNaseOUT Recombinant Ribonuclease Inhibitor, also included in the enzyme mix, is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation.

Reaction mix
The 2X RT reaction mix includes oligo(dT)₂₀, random hexamers, MgCl₂, and dNTPs in a buffer formulation that has been optimized for qRT-PCR. E. coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA:RNA hybrid molecule after first-strand synthesis. This has been shown to increase sensitivity in qRT-PCR.

Using SuperScript III First-Strand Synthesis SuperMix
This SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT-PCR, with a broad dynamic range that supports accurate quantification of high-copy mRNA from up to 1 μg of total RNA. Reagents are provided for 50 or 250 RT reactions of 20 μL each.

Order Info

Shipping Condition: Dry ice

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Specifications

• Content And Storage:

  • 2X RT Reaction Mix (2 x 1.25 mL)
  • RT Enzyme Mix (500 μL)
  • E. coli RNase (250 μL)
  
  Sufficient reagents for 250 reactions at 20 μL reaction volumes.
  Store at –20°C.
  
  

• Detection Method: qPCR
• Format: Super Mix
• GC-Rich PCR Performance: High
• Reaction Speed: 30 min.
• Technique: Reverse Transcription
• Optimal Reaction Temperature: 50°C
• Quantity: 250 rxns
• Reverse Transcriptase: SuperScript III
• Shipping Condition: Dry Ice
• For Use With (Application): Real Time PCR (qPCR)
• Final Product Type: First-Strand cDNA
• No. of Reactions: 250 Reactions
• Reaction Format: Master Mix
• Reagent Type: Reverse Transcription
• Starting Material: RNA

Frequently Asked Questions (FAQs)

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.

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.

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.

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 VILO kit is active at 42 degrees C due to the helper proteins.

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.