SuperScript IV CellsDirect cDNA Synthesis Kit

SuperScript IV CellsDirect cDNA Synthesis Kit is designed to synthesize first-strand cDNA directly from mammalian cell lysates without requiring the initial isolation of 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. This cDNA synthesis kit has been optimized for small cell samples, ranging from 10,000 cells down to a single cell.

Highlights

  • Time-saving–save up to two hours in overall workflow time
  • Streamlined workflow–lysis and RT reaction times reduced and completed in a single tube
  • Superior sensitivity–easy detection of low abundant targets
  • Direct convenience–no need to isolate RNA and store in your freezer
  • qPCR compatibility–enhanced performance with the new PowerTrack SYBR Green Master Mix

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Save time with our rapid protocol

SuperScript IV CellsDirect includes a powerful SuperScript IV reverse transcriptase enzyme and a simplified cDNA synthesis workflow that eliminates the need to purify RNA (Figure 1). With this basic approach in a single tube, you can avoid RNA loss and contamination by reducing the number of pipetting steps and manipulations. The end result is saving time and effort.

Figure 1. The SuperScript IV CellsDirect cDNA synthesis kit protocol goes from cells to cDNA synthesis in less than half the time that it takes to produce cDNA using traditional methods which include the need to isolate RNA. The key time savings steps are associated with RNA purification.

Superior 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 produces greater 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 2 and 3 illustrate the superior sensitivity and yield compared to the kits of competitors.

ACTB cDNA synthesis analyzed with a SYBR green assay

Figure 2. cDNA synthesis of ACTB analyzed using a SYBR Green assay. Using a serial dilution of HeLa S3 cells representing a range from 10 to 10,000 cells per sample, cells were lysed and cDNA synthesized according to each kit manufacturer’s specifications. Analysis was performed using the PowerTrack SYBR Green Master Mix.
Bar graph showing a comparison of the CT difference, which represents the yield. Graph uses the same data from the results presented above demonstrating sensitivity and yield for high abundance transcripts (ACTB)

Figure 3. Bar graph showing a comparison of the CT difference, which represents the yield. Graph uses the same data from the results presented above demonstrating sensitivity and yield for high abundance transcripts (ACTB).

To demonstrate sensitivity and yield for low abundance targets, the SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from BCL2 using target specific primers. Figure 4 and 5 illustrate the superior 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.

BCL2 cDNA synthesis analyzed with a SYBR green assay

Figure 4. cDNA synthesis of BCL2 and analyzed using a SYBR Green assay. Using a serial dilution of HeLa S3 cells representing a range from 10 to 10,000 cells per sample, cells were lysed and cDNA synthesized according to each kit manufacturer’s specifications. Analysis was performed using the PowerTrack SYBR Green Master Mix.

Bar graph using the same data as above showing the product vs. competitor kits across dilution series demonstrating sensitivity and yield for low abundance transcripts (BCL2)

Figure 5. Bar graph using the same data as above showing the product vs. competitor kits across dilution series demonstrating sensitivity and yield for low abundance transcripts (BCL2).

To further demonstrate sensitivity and yield, SuperScript IV CellsDirect cDNA synthesis kit was used to synthesize cDNA from PGK1, a moderately abundant gene target, using target specific primers. Figure 6 and 7 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.

PGK1 cDNA synthesis analyzed with a TaqMan assay

Figure 6. cDNA synthesis of PGK1 and analysis using a TaqMan assay. Using a serial 10-fold dilution of HeLa S3 cells representing a sample sizes from 10 to 10,000 cells per sample, cells were lysed and cDNA synthesized according to each kit manufacturer’s specifications. Analysis was performed using the TaqMan Fast Advanced Master Mix.
Bar graph uses the same data as above showing the product vs competitor kits across dilution series demonstrating sensitivity and yield for moderately abundant transcripts (PGK1)

Figure 7. Bar graph uses the same data as above showing the product vs competitor kits across dilution series demonstrating sensitivity and yield for moderately abundant transcripts (PGK1).

The SuperScript IV CellsDirect cDNA synthesis kit consistently outperformed competitors for a range of low to high abundant genes across many cell lines including Induced Pluripotent Stem Cells (iPSC).

Figure 8. Higher reverse transcriptase efficiency with SuperScript IV CellsDirect cDNA synthesis Kit than with other commercial kits (SuperScript III CellsDirect cDNA synthesis Kit (Invitrogen), SingleShot Probes Kit (Bio-Rad), FastLane Cell cDNA Kit (Qiagen),  RealTime ready Cell Lysis Kit + Transcriptor First Strand cDNA Synthesis Kit (Roche). cDNA synthesis was performed from 1000 human iPSC stem cells inputs to lysis and analyzed with various from low to high expression gene primers in TaqMan assay.

The SuperScript IV CellsDirect cDNA synthesis kit consistently outperformed competitors during linearity tests.

Amplification plot of four experiments showing the ten-fold dilution series of starting cells inputs (10000 - 1) between competitors. Figure shows that SuperScript IV CellsDirect has the best linearity compared to other commercial kits

Figure 9. Better linearity with SuperScript IV CellsDirect cDNA synthesis Kit than with other commercial kits between the diluted sample sequence in qPCR assay.

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