Direct reverse transcription kits are designed to synthesize cDNA directly from cell lysates without isolating RNA. Lysis and reverse transcription are performed in the same tube. The resulting first-strand cDNA is ready to use in downstream applications including PCR, qPCR, and NGS.


Cells-to-cDNA kits: Selection guide


Table 1: Comparison of cells-to-cDNA kits

 

Photo of product package and multiple vials with kit reagents

SuperScript IV CellsDirect cDNA Synthesis Kit

Photo of product package and multiple vials with kit reagents

SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit

Intended useFirst-strand cDNA synthesis directly from mammalian cell lysatesTemplate switching reverse transcription and global preamplification of full-length cDNA
RNA purification requiredNoNo
Starting material1–10,000 cells1–1,000 cells
2 pg–10 ng total RNA
Final reaction productcDNAPCR amplified cDNA
WorkflowWorkflow schematic with three steps to cDNA synthesisWorkflow schematic with four steps to preamplification
Reagents includedLysis → RTLysis → RT → PreAmp
RT primerRandom + oligo(dT)Oligo(dT)-based
DNA removalYes (DNase digestion)Yes (mRNA enrichment)
Number of targetsLimitedFull transcriptome
Sensitivity++++
Yield++++
ApplicationsRT-PCR, RT-qPCR, cloningGlobal preamplification, RNA-seq, RT-qPCR
 Order nowOrder now

The cDNA synthesis master mix from the SuperScript IV Single Cell/Low-Input cDNA PreAmp kit is now available as a standalone SuperScript IV Template Switching RT Master Mix.

This master mix is designed for high template switching efficiency in cDNA synthesis reactions and contains SuperScript IV Reverse Transcriptase, RNase Inhibitor, and all other reverse transcription reaction components in a convenient premixed format. Only RNA template, RT primer, and template switching oligo (TSO) of choice need to be added. The master mix is compatible with a wide range of total RNA (from 2 pg to 4 µg) and is supplied with lysis buffer to enable direct cDNA synthesis from intact mammalian cells (1–1,000 cells per sample).

In conjunction with SuperScript IV Template Switching RT Master Mix, we offer Template Switching Oligo and Capturing Oligo (dT) Primer, both containing adaptor sequences complementary to the Preamplification Primer.

Photo of product package and three vials with kit reagents.

SuperScript IV template switching RT Master Mix

Order now

Photo of product package and one vial with red cap

Template Switching Oligo

Order now

Photo of product package and one vial with purple cap

Preamplification Primer

Order now

Photo of product package and one vial with blue cap

Capturing oligo (dT) primer

Order now


How does the template-switching mechanism work?

In the template switching mechanism, a reverse transcriptase enzyme binds to the RNA template and RT primer duplex and begins synthesizing a complementary DNA strand. When the reverse transcriptase reaches the 5’ end of the RNA template, terminal deoxynucleotidyl transferase (TdT) activity adds 1–3 additional nucleotides to the cDNA end, enabling binding of the template switching oligo and incorporation of known sequence at the 3’ end of the resulting cDNA (Figure 1). This cDNA can be used for amplification by PCR, gene expression analysis by real-time PCR, or as a template for 5’RACE, RNA-seq, or second strand cDNA synthesis.


Advantages of SuperScript IV Single Cell/Low Input cDNA PreAmp kit

  • Streamlined and timesaving workflow—One tube protocol with short reaction times
  • Exceptional sensitivity—Easy detection of low-abundant targets from as low as a single cell or 2 pg of total RNA
  • High quality cDNA—Full-length transcript information with uniform coverage
  • Global cDNA pre-amplification—Compatible with downstream analysis by NGS or qPCR

 

The SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit workflow takes significantly less time when compared to workflows with competitor products (Figure 2).

Workflow schematic indicates that SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit has the shortest workflow time of 133 minutes compared to competitors workflow time ranging between 210 to 275 minutes.

Figure 2Workflow comparisons with competitors. Workflows and timelines for the SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit in comparison to the Smart-seq3 Protocol V.3 workflow, NEBNext™ kit workflow, and Takara SMART-Seq v4™.

Experimental data support that SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit generates high cDNA yields from low-input RNA or single-cell RNA. cDNA yields from low-input (2 pg) Universal Human Reference RNA (UHRR) were almost twice the yield from NEBNext™ Single Cell/Low Input cDNA Synthesis and Amplification Module (Figure 3).

SuperScript IV PreAmp kit uniformly preamplifies transcripts for reliable RNA sequencing. Libraries prepared from cDNA generated with the SuperScript IV PreAmp kit resulted in low bias across most of the normalized transcript distance (Figure 4).

The SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit workflow takes significantly less time when compared to workflows with competitor products (Figure 2).

Workflow schematic indicates that SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit has the shortest workflow time of 133 minutes compared to competitors workflow time ranging between 210 to 275 minutes.

Figure 2Workflow comparisons with competitors. Workflows and timelines for the SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit in comparison to the Smart-seq3 Protocol V.3 workflow, NEBNext™ kit workflow, and Takara SMART-Seq v4™.

Experimental data support that SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit generates high cDNA yields from low-input RNA or single-cell RNA. cDNA yields from low-input (2 pg) Universal Human Reference RNA (UHRR) were almost twice the yield from NEBNext™ Single Cell/Low Input cDNA Synthesis and Amplification Module (Figure 3).

SuperScript IV PreAmp kit uniformly preamplifies transcripts for reliable RNA sequencing. Libraries prepared from cDNA generated with the SuperScript IV PreAmp kit resulted in low bias across most of the normalized transcript distance (Figure 4).


Advantages of SuperScript IV CellsDirect cDNA Synthesis Kit

  • Streamlined and timesaving workflow—Lysis and RT reaction completed in a single tube; saves up to two hours in overall workflow time
  • Convenience—No separate step to isolate RNA
  • qPCR compatibility—Enhanced performance with the new PowerTrack SYBR Green Master Mix
  • Exceptional sensitivity—Easy detection of low abundance targets, ranging from 10,000 cells to a single cell

SuperScript IV CellsDirect cDNA synthesis kit requires a simplified cDNA synthesis workflow that eliminates the need to purify RNA (Figure 5). With this approach, fewer pipetting steps are required, which helps prevent RNA loss and contamination, saving time and effort. In addition, SuperScript IV CellsDirect cDNA Synthesis Kit delivers exceptional sensitivity, yield, and linearity.

Workflow schematics show significant time gains, of approximately 50 minutes, using SuperScript IV CellsDirect cDNA synthesis kit when compared to traditional cDNA synthesis methods.

Figure 5. Comparison workflow versus traditional methods. The protocol used for SuperScript IV CellsDirect cDNA synthesis kit goes from cells-to-cDNA in less than half the time it takes to produce cDNA using traditional methods; the time savings are associated with RNA purification.

The SuperScript IV CellsDirect cDNA synthesis kit is sensitive across a range of cell types and sample sizes. It consistently outperforms kits from competitors by producing higher yields for abundant (ACTB), moderately abundant (PGK1), and low abundance (BCL2) transcripts (Figure 6).

The SuperScript IV CellsDirect kit demonstrated the strongest linear correlation across four mRNA targets (ACTB, BCL2, PGK1, PPIA) across serial dilution of HeLa S3 cells ranging from 1 to 10,000 cells (Figure 7).

Line plots with Ct values and cell inputs
Figure 7. The SuperScript IV CellsDirect kit demonstrates linearity across a wide dynamic range of cellular inputs, and superior RNA detection sensitivity.

SuperScript IV CellsDirect cDNA synthesis kit requires a simplified cDNA synthesis workflow that eliminates the need to purify RNA (Figure 5). With this approach, fewer pipetting steps are required, which helps prevent RNA loss and contamination, saving time and effort. In addition, SuperScript IV CellsDirect cDNA Synthesis Kit delivers exceptional sensitivity, yield, and linearity.

Workflow schematics show significant time gains, of approximately 50 minutes, using SuperScript IV CellsDirect cDNA synthesis kit when compared to traditional cDNA synthesis methods.

Figure 5. Comparison workflow versus traditional methods. The protocol used for SuperScript IV CellsDirect cDNA synthesis kit goes from cells-to-cDNA in less than half the time it takes to produce cDNA using traditional methods; the time savings are associated with RNA purification.

The SuperScript IV CellsDirect cDNA synthesis kit is sensitive across a range of cell types and sample sizes. It consistently outperforms kits from competitors by producing higher yields for abundant (ACTB), moderately abundant (PGK1), and low abundance (BCL2) transcripts (Figure 6).

The SuperScript IV CellsDirect kit demonstrated the strongest linear correlation across four mRNA targets (ACTB, BCL2, PGK1, PPIA) across serial dilution of HeLa S3 cells ranging from 1 to 10,000 cells (Figure 7).

Line plots with Ct values and cell inputs
Figure 7. The SuperScript IV CellsDirect kit demonstrates linearity across a wide dynamic range of cellular inputs, and superior RNA detection sensitivity.


Cells-to-cDNA kits

Not sure if direct or template switching reverse transcription is right for you? Use our selection tool to find products fit for your cDNA synthesis experiments.


Resources for direct reverse transcription from cells-to-cDNA

Resources

Reverse transcriptase selection tool
Find the right products to fit your cDNA synthesis needs.

Reverse transcriptase webinars
Stay up-to-date on current industry trends in cDNA synthesis applications with on-demand webinars.

Reverse transcriptase literature
Access white papers and technical and application notes for insights on cutting edge reverse transcription technology.

Reverse transcriptase videos
Watch instructional videos on troubleshooting tips, how-to videos, and explainers related to cDNA synthesis.

Reverse transcriptase education
Review educational resources on topics including reverse transcription basics, cDNA synthesis, enzyme selection, troubleshooting tips, and cDNA applications in molecular biology.

Support

PCR and cDNA Synthesis Support Center
Find support for every step of your cDNA synthesis experiments.

Tm calculator
Handy tool to calculate Tm (melting temperature) of primers and annealing temperatures.

Contact us
Get in touch with our technical application scientists for questions regarding reverse transcription enzymes, master mixes, and cDNA synthesis kits.

Mobile and desktop applications
Access content and helpful tools through SmartIST and Cloning Bench mobile apps.

OEM and custom solutions
Source reverse transcriptase and other molecular biology enzymes, PCR reagents, and plastics customized to your needs.

仅供科研使用,不可用于诊断目的。