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Direct Reverse Transcription: From Cells-to-cDNA |
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
|
| |
| Intended use | First-strand cDNA synthesis directly from mammalian cell lysates | Template switching reverse transcription and global preamplification of full-length cDNA |
| RNA purification required | No | No |
| Starting material | 1–10,000 cells | 1–1,000 cells 2 pg–10 ng total RNA |
| Final reaction product | cDNA | PCR amplified cDNA |
| Workflow |  |  |
| Reagents included | Lysis → RT | Lysis → RT → PreAmp |
| RT primer | Random + oligo(dT) | Oligo(dT)-based |
| DNA removal | Yes (DNase digestion) | Yes (mRNA enrichment) |
| Number of targets | Limited | Full transcriptome |
| Sensitivity | + | +++ |
| Yield | + | +++ |
| Applications | RT-PCR, RT-qPCR, cloning | Global preamplification, RNA-seq, RT-qPCR |
| Order now | Order 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.
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.
Click image to enlarge
Figure 1: Schematic representation of template switching reverse transcription and cDNA amplification.
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).
Figure 2. Workflow 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).
Click image to enlarge
Figure 3. Comparison of cDNA yields from SuperScript IV PreAmp kit with NEBNext™ preamplification kit. SuperScript IV PreAmp kit produces excellent cDNA yield, compared to the NEBNext™ Single Cell/Low Input cDNA Synthesis and Amplification Module. Error bars represent standard deviation among at least five replicates.
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).
Click image to enlarge
Figure 4. Normalized transcript coverage across several different input types. Single-cell RNA was reverse transcribed and the obtained cDNA was preamplified using the recommended number of PCR cycles. The Invitrogen Collibri ES DNA Library Prep Kit for Illumina™ Systems with UD indexes was used to prepare libraries for sequencing using the MiSeq™ Sequencing system.
The SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit workflow takes significantly less time when compared to workflows with competitor products (Figure 2).
Figure 2. Workflow 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).
Click image to enlarge
Figure 3. Comparison of cDNA yields from SuperScript IV PreAmp kit with NEBNext™ preamplification kit. SuperScript IV PreAmp kit produces excellent cDNA yield, compared to the NEBNext™ Single Cell/Low Input cDNA Synthesis and Amplification Module. Error bars represent standard deviation among at least five replicates.
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).
Click image to enlarge
Figure 4. Normalized transcript coverage across several different input types. Single-cell RNA was reverse transcribed and the obtained cDNA was preamplified using the recommended number of PCR cycles. The Invitrogen Collibri ES DNA Library Prep Kit for Illumina™ Systems with UD indexes was used to prepare libraries for sequencing using the MiSeq™ Sequencing system.
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.
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).
Click image to enlarge
Figure 6.Comparative cDNA yields among direct RT kits for a variety of cellular transcripts. Samples of three distinct mammalian cell types (Jurkat, K562, and iPSCs) were used for cDNA generation using the SuperScript IV CellsDirect kit and four other commercially available direct RT kits. qPCR was performed to evaluate cDNA yields from 44 different RNA transcripts, representing a wide range of cellular abundance. The SuperScript IV CellsDirect kit had the highest cDNA yields
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).
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.
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).
Click image to enlarge
Figure 6.Comparative cDNA yields among direct RT kits for a variety of cellular transcripts. Samples of three distinct mammalian cell types (Jurkat, K562, and iPSCs) were used for cDNA generation using the SuperScript IV CellsDirect kit and four other commercially available direct RT kits. qPCR was performed to evaluate cDNA yields from 44 different RNA transcripts, representing a wide range of cellular abundance. The SuperScript IV CellsDirect kit had the highest cDNA yields
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).
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
SuperScript IV CellsDirect cDNA Synthesis Kit
SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit
SuperScript IV Template-Switching RT Master Mix
Accessory products
Reverse transcriptases: Selection tool
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