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  7. Invitrogen™ SuperScript™ IV Single Cell/Low Input cDNA PreAmp Kit

Invitrogen™ SuperScript™ IV Single Cell/Low Input cDNA PreAmp Kit

Product Code. 17473802
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Product Code. 17473802 Supplier Invitrogen™ Supplier No. 11752048

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Description

The Invitrogen SuperScript IV Single Cell/Low Input cDNA PreAmp Kit is designed for efficient cDNA synthesis and amplification directly from intact single cells (1–1,000) or low amounts of total RNA (2 pg–10 ng).

The Invitrogen SuperScript IV Single Cell/Low Input cDNA PreAmp Kit is designed for efficient cDNA synthesis and amplification directly from intact single cells (1–1,000) or low amounts of total RNA (2 pg–10 ng). It contains all required components to perform cell lysis, reverse transcription (RT), and PCR amplification in a convenient premixed format. A combination of superior enzymes (SuperScript IV Reverse Transcriptase and Platinum SuperFi U DNA Polymerase) enables high yields, sensitivity, and accuracy. Due to the short reaction times and minimal hands-on time, this simple one-tube preamplification protocol can be completed in just over two hours. The high quality global cDNA obtained can be used for next-generation sequencing (NGS) applications or for gene expression analysis by real-time PCR.

Features of the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit include:
• Superior sensitivity for easy detection of low abundant targets (as low as single cell or 2 pg total RNA)
• High quality cDNA for full-length transcript information with uniform coverage
• Streamlined workflow with simple one-tube protocol
• Short lysis and reverse transcription times for time savings
• Global preamplification compatible with downstream analysis by NGS or real-time PCR

The SuperScript IV Single Cell/Low Input cDNA PreAmp Kit leverages the terminal deoxynucleotidyl transferase (TdT) activity of the SuperScript IV Reverse Transcriptase. Capturing oligonucleotide containing oligo(dT) and adapter sequences is used as primer for selective first strand cDNA synthesis from poly(A)-containing RNAs. When the reverse transcriptase reaches the 5‘ end of the RNA template, TdT activity adds 1–3 extra nucleotides to the cDNA end, enabling template switching and ligation-free incorporation of adapter sequence in the 3‘ end of the resulting cDNA. The high sensitivity and template switching efficiency of SuperScript IV Reverse Transcriptase allow the capture of even low-abundance targets.

Adapter sequences incorporated in both ends of the cDNA serve as primer-binding sites in the subsequent PCR amplification step, allowing global preamplification of the full-length templates. The included Platinum SuperFi U Preamplification Master Mix contains a superior high-fidelity DNA polymerase that ensures efficient amplification of long templates (up to 10 kb) with no PCR-induced bias or errors.

TRUSTED_SUSTAINABILITY

Specifications

Content And Storage

• 10X Lysis Buffer (48 μL)
• RNase Inhibitor (19 μL)
• Capturing Oligonucleotide (48 μL)
• 4X SuperScript IV cDNA Synthesis Master Mix (240 μL)
• Template Switching Oligonucleotide (48 μL)
• 2X Platinum SuperFi U Preamplification Master Mix (2 x 1.2 mL)
• Preamplification primer (48 μL)
• Nuclease-free water (2 x 1.25 mL)

Store at –15 to –25°C.
Format Kit
GC-Rich PCR Performance High
Polymerase Platinum SuperFi U DNA Polymerase
Reaction Speed 30 min.
Technique Reverse Transcription, Preamplification
Optimal Reaction Temperature 50°C
Quantity 48 reactions
Reverse Transcriptase SuperScript IV
Shipping Condition Dry Ice
For Use With (Application) Next-Generation Sequencing (NGS), Real Time PCR (qPCR)
Final Product Type PCR Amplified cDNA
No. of Reactions 48 Reactions
Reagent Type Reverse Transcription
Size (Final Product) Up to 10 kb
Starting Material Cells or RNA
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Frequently Asked Questions (FAQs)

What products do you recommend for downstream analysis by qPCR when using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

We recommend using TaqMan Fast Advanced Master Mix for all TaqMan‑based detection methods.

We recommend using PowerTrack SYBR Green Master Mix for all SYBR Green‑based detection methods.
How many preamplification cycles are required when using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

Cycle number depends on sample type and input amount.
For example:
- 21 cycles for 2 pg of total RNA
- 18 cycles for 10 pg of total RNA, or single cells
- 15 cycles for 100 pg of total RNA, or up to 10 cells
- 11 cycles for 1-10 ng of total RNA, or 100–1,000 cells
- RNA quantity in cells can vary by cell type, cell cycle, and cell health. Optimization of the PCR cycle number may be needed to reach desired yields. An additional 2–3 cycles are required for smaller cells, such as Jurkat, Daudi, or peripheral blood mononuclear cells (PBMC).
What enzyme is included in Platinum SuperFi U Preamplification Master Mix that is a component of the SuperScript IV Sincle Cell/Low Input cDNA PreAmp Kit?

The master mix includes Platinum SuperFi U DNA Polymerase. It provides the same benefits as Platinum SuperFi II DNA Polymerase, but is also compatible with uracil.
Is purification of total RNA required when using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

Lysis buffer is included in the kit, therefore intact mammalian cells can be used. Purification of total RNA is required for cDNA preamplification of RNA isolated from plant, fungi, or other cell wall‑containing organisms.
Is rRNA depletion or mRNA enrichment required when using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

rRNA depletion or mRNA enrichment is not required, because only poly(A)‑containing RNA is amplified.
Can I use different magnetic beads, rather than the recommended Agencourt AMPure XP beads, with the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

The SuperScript IVSingle Cell/Low Input cDNA PreAmp Kt has only been tested with Agencourt AMPure XP beads. Therefore, we cannot recommend any alternatives.

What do you recommend for sample cleanup when using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

We recommend using Agencourt AMPure XP beads (Beckman Coulter A63880) according to the protocol provided in the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit’s User Guide.
When using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit, is the purification step necessary after preamplification?

For downstream analysis by next-generation sequencing (NGS), preamplified cDNA should be purified to remove residual primers, nucleotides, and enzymes that may impact library preparation. For product analysis by qPCR, purification is not required but can be used to concentrate samples for detection of low-expression targets. For unpurified cDNA samples, samples should be diluted 1:10 or 1:100, prior to qPCR. For purified cDNA samples, dilution is not necessary. The purification step is also recommended prior to storing preamplified cDNA at -20 degrees C for more than 1 week.
How does the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit differ from TaqMan PreAmp Master Mix?

TaqMan PreAmp Master Mix is designed for targeted preamplification of up to 100 targets from small amounts of cDNA, while SuperScript IV Single Cell/Low Input cDNA PreAmp Kit allows global preamplification of full-length cDNA from single cells or low input RNA. It provides reagents for cell lysis, reverse transcription and preamplification steps.
Is the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit compatible with targeted preamplification?

This kit is designed for global full-length cDNA synthesis and preamplification. However, in downstream analysis, either whole transcriptome or specific targets of interest can be analyzed. It is compatible with downstream analysis by both next-generation sequencing (NGS) and real-time PCR.
Does the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit work with degraded and/or FPPE samples?

This kit is designed for global full-length cDNA preamplification. It leverages SMART technology utilizing oligo (dT)-based priming. Therefore, to obtain full length cDNA, we recommend high quality intact poly(A) RNA.

In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will ezDNase be active to affect RNA or the RT reaction?

The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50 degrees C). The additional inactivation step is therefore not required for most RT-qPCR applications. If the RNA sample is to be used for RT-PCR of fragments ≥3 kb, incubate the sample for 5 minutes at 55 degrees C in the presence of 10 mM DTT to inactivate the enzyme.

Which SuperScript IV RT format do you recommend for real-time PCR applications?

For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix. The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps and therefore reduces variation in the data. SuperScript IV RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.
Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?

The only difference is that the incubation time for the reverse transcription reaction has been reduced to 10 minutes and inactivation time has been reduced to 5 minutes at 85 degrees C.
Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?

When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density).

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

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