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SunScript™ Reverse Transcriptase

Basis of the Reverse Transcription Reaction

Basis of the Reverse Transcription Reaction


 SunScript™ Reverse Transcriptase allows reaction temperatures up to 85°C

SunScript™ Reverse Transcriptase allows reaction temperatures up to 85°C

100 ng of total RNA from HeLa cells were reverse transcribed at the indicated temperatures for 60 min with SunScript™ Reverse Transcriptases and Oligo(dT)18 primer, followed by inactivation at 95°C for 10 min. Presence of GAPDH cDNA was detected by PCR (product length 305 bp).
High reaction temperatures are advantageous to transcribe RNAs with complicated secondary structures.


 

SunScript™ Reverse Transcriptase: High sensitivity over a wide range of RNA concentrations

SunScript™ Reverse Transcriptase: High sensitivity over a wide range of RNA concentrations

Sensitivity test: RT reactions were performed at 70°C for 60 min. Inactivation at 95°C for 10 min.

Primer set : Human GAPDH305bp set

1- 1 µg Human total RNA from HeLa cells
2- 100 ng Human total RNA from HeLa cells
3- 10 ng Human total RNA from HeLa cells
4- 1 ng Human total RNA from HeLa cells
5- 100 pg Human total RNA from HeLa cells


SunScript™ Reverse Transcriptase: Transcription of long mRNAs

SunScript™ Reverse Transcriptase: Transcription of long mRNAs

1- SunScript™ RT RNaseH+ replicate 1
2- SunScript™ RT RNaseH+ replicate 2
3- SunScript™ RT RNaseH+ replicate 3
4- SunScript™ RT RNaseH- replicate 1
5- SunScript™ RT RNaseH- replicate 2
6- SunScript™ RT RNaseH- replicate 3

Assay for long mRNA reverse transcription: 3 independent RT reactions were performed at 65°C for 60 min using Oligo(dT)20 primers. Inactivation at 95°C for 10 min. PCR Primer sets : mmNEB (>16kb from 3’end of mRNA; mRNA total length 22,5kb); mmDnah8 (> 8,5kb from 3’end of mRNA; mRNA total length 14,5kb)

 


 

SunScript™ Reverse Transcriptase: high cDNA synthesis rate

SunScript™ Reverse Transcriptase: high cDNA synthesis rate

1 µg of total RNA from HeLa cells was reverse transcribed at 65°C for the indicated times with SunScript™ Reverse Transcriptases and Oligo(dT)18 primer, followed by inactivation at 95°C 10 min.
PCR Primer set: hsHERC4033 (>11kb from 3’end of mRNA; mRNA total length 15.3kb)

 Reverse Transcriptases


 

SunScript™ One Step RT-PCR

Overview over the One Step RT-PCT reaction

Overview over the One Step RT-PCT reaction


SunScript™ One Step RT-PCR kit detects genes with different expression levels

SunScript™ One Step RT-PCR kit detects genes with different expression levels

100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for the indicated genes (GAPDH, HERC GUSB, CLTC and ACTIN).
The kit easily detects genes with vastly different expression levels.


SunScript™ One Step RT-PCR kit performs the RT reaction up to temperatures of 85ºC

SunScript™ One Step RT-PCR kit performs the RT reaction up to temperatures of 85ºC

100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for the GAPDH open reading frame (product length 305 bp).
The RT reaction can be performed at temperatures up to 85ºC, making this the best choice for detecting difficult RNAs, with high degrees of secondary structure.


 

SunScript™ One Step RT-PCR kit sensitivity

SunScript™ One Step RT-PCR kit sensitivity

The indicated amounts of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for GAPDH cDNA (product length 305 bp).
The sensitivity of the kit is good enough to detect expressed genes from total RNA starting material in the order of picograms.


 

SunScript™ One Step RT-PCR kit produces highly reproducible results

SunScript™ One Step RT-PCR kit produces highly reproducible results

100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for the indicated genes (4 replicates are performed per gene).
The four independent replicates per gene show similar sensitivity and yield, making this kit the best choice for processing multiple samples with minimum deviations.


 

SunScript™ One Step RT-PCR kit: amplification of difficult RNAs 

SunScript™ One Step RT-PCR kit: amplification of difficult RNAs

100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ RT-PCR Kit and four different specific primer pairs for a fragment of the TAFII100 cDNA with 75% GC content (product lengths 430-470 bp).
The RT-PCR protocol with sample denaturation was performed here and the cDNA synthesis step was performed at 60ºC.
The four oligo pairs produced effective amplification bands despite the elevated GC content of the template RNA.


 

SunScript™ One Step RT-qPCR

Suggested protocols: Standard and fast

Suggested protocols: Standard and fast


 

SunScript™ One Step RT-qPCR kit easily detects low abundance gene products and outperforms high-temperature one step RT-qPCR competitors

One Step RT-qPCR kit easily detects low abundance gene products and outperforms high-temperature one step RT-qPCR competitors

Figure 1: Comparison of amplification of the low abundance transcripts bax and herc2 at 50°C and 60°C reverse transcription reaction temperature. RNA-source: HEK-293 total RNA 100 ng. Light blue: SYGNIS SunScript™ One Step RT-qPCR kit, dark blue, red, orange: Competitor kits.


SunScript™ One Step RT-qPCR kit has a broad dynamic range of quantification

SunScript™ One Step RT-qPCR kit has a broad dynamic range of quantification

Figure 2: Comparison of amplification of three gene products strongly diverging in their expression profiles (β-actin, bax, herc2) at 50°C and 60°C reverse transcription reaction temperature. RNA-source: HEK-293 total RNA 100 ng. Legend gives FPKM values for the transcripts in this cell line (RNAseq; www.proteinatlas.org).


 

SunScript™ One Step RT-qPCR kit faithfully quantifies dilution series for high quality standard curves

SunScript™ One Step RT-qPCR kit faithfully quantifies dilution series for high quality standard curves

Figure 3: β-actin quantification in a log10-dilution series of total RNA from HEK-293 cells. RT reaction performed at 60°C. Note the excellent log-linearity of the standard curve.


 

SunScript™ One Step RT-qPCR shows excellent reproducibility

SunScript™ One Step RT-qPCR shows excellent reproducibility

Figure 4: β-actin quantification in a log10-dilution series of total RNA from HEK-293 cells. RT reaction was performed at 60°C. Seven independent replicates for each dilution shown in the Amplification graph visualize the high reproducibility of the reaction. The bar graph on the right depicts the log-linearity of the RT-qPCR performance in this assay (Cq values shown). Note the small standard deviations (SD) in the bar graph.


 

SunScript™ One Step RT-qPCR kit works up to RT reaction temperatures of at least 75°C

SunScript™ One Step RT-qPCR kit works up to RT reaction temperatures of at least 75°C

Figure 5: Comparison of RT step reaction temperatures for 3 gene products with widely differing abundance levels. Input: HEK-293 cell total RNA (100 ng). Amplification products are obtained up to an RT reaction temperature of up to 75°C without problems. Optimal amplification occurs around 60°C, with primers having a calculated melting temperature (TM) of 55-60°.