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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 and qPCR. Cells-to-cDNA kit options are:
SuperScript IV Single Cell/Low-Input
cDNA PreAmp Kit
A combination of SuperScript IV and Platinum SuperFi enzymes along with template switching technology allows full length cDNA amplification with high yields, exceptional sensitivity, and accuracy for downstream analysis by NGS or real-time PCR.
SuperScript IV CellsDirect
cDNA Synthesis Kit
The SuperScript IV CellsDirect cDNA Synthesis Kit is designed for first-strand cDNA synthesis directly from mammalian cell lysates and provides reagents for cell lysis and reverse transcription.
This kit includes pre-mixed components containing the SuperScript IV Reverse Transcriptase. The simplified cDNA synthesis workflow eliminates the need to purify RNA.
Advantages of SuperScript IV Single Cell/Low-Input cDNA PreAmp kit
- Streamlined workflow and timesaving—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
- Universal—global cDNA pre-amplification compatible with downstream analysis by NGS or qPCR
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 1).
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Figure 1. 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.
The SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit workflow takes significantly less time when compared to the Smart-seq3 Protocol V.3 workflow and the NEBNext™ kit workflow (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 and the NEBNext™ kit workflow.
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 1).
Click image to enlarge
Figure 1. 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.
The SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit workflow takes significantly less time when compared to the Smart-seq3 Protocol V.3 workflow and the NEBNext™ kit workflow (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 and the NEBNext™ kit workflow.
Advantages of SuperScript IV CellsDirect cDNA Synthesis kit
- Streamlined workflow and timesaving—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
Short workflow
SuperScript IV CellsDirect cDNA synthesis kit requires a simplified cDNA synthesis workflow that eliminates the need to purify RNA (Figure 3). With this approach, fewer pipetting steps are required, which helps prevent RNA loss and contamination hence saving time and effort.
Figure 3. 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.
Exceptional sensitivity and yield
The SuperScript IV CellsDirect cDNA synthesis kit consistently outperforms kits from competitors. It is sensitive across a range of cell numbers and sample sizes and can produce higher yields for abundant (ACTB), moderately abundant (PGK1), and low abundance (BCL2) transcripts in studies with HeLa S3 cells.
The SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from ACTB, an abundant transcript, using target specific primers. Figure 4A and 4B illustrate the exceptional sensitivity and yield compared to commercially available kits from competitors.
Click image to enlarge
Click image to enlarge
Figure 4. Comparison of cDNA synthesis kits to synthesize cDNA from ACTB, a high abundance transcript. (A) cDNA synthesis of ACTB analyzed using SYBR Green assay: serially diluted HeLa S3 cells, ranging between 10 to 10,000 cells per sample, were lysed and cDNA was synthesized according to each kit manufacturer’s specifications. Real-time PCR analysis was performed using the PowerTrack SYBR Green Master Mix. (B) Bar graph showing a comparison of the Ct difference, which represents the yield. SuperScript IV CellsDirect cDNA synthesis kit consistently has high cDNA yields when compared to competitor products.
The SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from PGK1, a moderately abundant gene target, using target specific primers. Figures 5A and 5B illustrate sensitivity and yield across a 10-fold dilution series of HeLa S3 cells (10 to 10,000 cells) as compared to the kits of competitors.
Click image to enlarge
Click image to enlarge
Figure 5. Comparison of cDNA synthesis kits to synthesize cDNA from PGK1, a moderate abundance transcript. (A) cDNA synthesis of PGK1 and amplification plot using a TaqMan assay: serially diluted HeLa S3 cells were lysed and cDNA was synthesized according to each kit manufacturer’s specifications. Analysis was performed using the TaqMan Fast Advanced Master Mix. (B) Bar graph demonstrating sensitivity and yield for moderately abundant transcript, PGK1. SuperScript IV CellsDirect cDNA synthesis kit is the top performer.
The SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from BCL2, a low abundance target, using target specific primers. Figure 6A and 6B illustrate the exceptional sensitivity and yield across the full 10-fold dilution series of HeLa S3 cells (10 to 10,000 cells) as compared to the kits of competitors.
Click image to enlarge
Click image to enlarge
Figure 6. Comparison of cDNA synthesis kits to synthesize cDNA from BCL2, a low abundance transcript. (A) cDNA synthesis of BCL2 and analyzed using a SYBR Green assay: serially diluted HeLa S3 cells, were lysed, and cDNA was synthesized according to manufacturer specifications. Analysis was performed using the PowerTrack SYBR Green Master Mix. (B)Bar graph demonstrating sensitivity and yield for low abundance transcript, BCL2. SuperScript IV CellsDirect cDNA synthesis kit is the top performer.
The SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from ACTB, an abundant transcript, using target specific primers. Figure 4A and 4B illustrate the exceptional sensitivity and yield compared to commercially available kits from competitors.
Click image to enlarge
Click image to enlarge
Figure 4. Comparison of cDNA synthesis kits to synthesize cDNA from ACTB, a high abundance transcript. (A) cDNA synthesis of ACTB analyzed using SYBR Green assay: serially diluted HeLa S3 cells, ranging between 10 to 10,000 cells per sample, were lysed and cDNA was synthesized according to each kit manufacturer’s specifications. Real-time PCR analysis was performed using the PowerTrack SYBR Green Master Mix. (B) Bar graph showing a comparison of the Ct difference, which represents the yield. SuperScript IV CellsDirect cDNA synthesis kit consistently has high cDNA yields when compared to competitor products.
The SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from PGK1, a moderately abundant gene target, using target specific primers. Figures 5A and 5B illustrate sensitivity and yield across a 10-fold dilution series of HeLa S3 cells (10 to 10,000 cells) as compared to the kits of competitors.
Click image to enlarge
Click image to enlarge
Figure 5. Comparison of cDNA synthesis kits to synthesize cDNA from PGK1, a moderate abundance transcript. (A) cDNA synthesis of PGK1 and amplification plot using a TaqMan assay: serially diluted HeLa S3 cells were lysed and cDNA was synthesized according to each kit manufacturer’s specifications. Analysis was performed using the TaqMan Fast Advanced Master Mix. (B) Bar graph demonstrating sensitivity and yield for moderately abundant transcript, PGK1. SuperScript IV CellsDirect cDNA synthesis kit is the top performer.
The SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from BCL2, a low abundance target, using target specific primers. Figure 6A and 6B illustrate the exceptional sensitivity and yield across the full 10-fold dilution series of HeLa S3 cells (10 to 10,000 cells) as compared to the kits of competitors.
Click image to enlarge
Click image to enlarge
Figure 6. Comparison of cDNA synthesis kits to synthesize cDNA from BCL2, a low abundance transcript. (A) cDNA synthesis of BCL2 and analyzed using a SYBR Green assay: serially diluted HeLa S3 cells, were lysed, and cDNA was synthesized according to manufacturer specifications. Analysis was performed using the PowerTrack SYBR Green Master Mix. (B)Bar graph demonstrating sensitivity and yield for low abundance transcript, BCL2. SuperScript IV CellsDirect cDNA synthesis kit is the top performer.
Cells-to-cDNA products
- SuperScript IV Single Cell/Low Input cDNA PreAmp Kit
- SuperScript IV CellsDirect cDNA Synthesis Kit
- SuperScript IV CellsDirect Lysis Reagents
- Cells-to-CT Kits
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