A novel high-temperature One Step RT-PCR kit based on the combination of SunScript™ Reverse Transcriptase RNaseH- and high-quality Taq Polymerase for the reverse transcription and PCR amplification of specific RNA fragments in one convenient single step.

• Improved thermal stability for the reverse transcription step:Works up to 85°C (60-65°C recommended).
• Transcription and amplification of difficult RNAs: Transcribes RNAs with high GC content or complex secondary structures.
• Great sensitivity:Starts with picogram amounts of RNA
• Improved PCR specificity: Hot start Taq polymerase greatly improves specificity during amplification
• High yields of amplified DNA
• High reproducibility across reactions:Best choice for processing multiple samples with minimum deviations.
• Easy handling:Reagents are added in a single step, which also minimizes dangers of contamination and reduces overall handling time.

The SunScript™ One Step RT-PCR Kit is an easy and reliable system designed for fast, specific and sensitive end point RT-PCR reactions. This kit contains all the components needed to perform both reverse transcription and PCR amplification in a single tube by using gene specific primers in a “one step” reaction. This minimizes contaminations and constitutes a convenient system to process multiple samples.

• One Step RT-PCR Protocol Overview
• Detection of mRNAs with different expression levels
• High thermal activity of the RT step
• High sensitivity
• High reproducibility
• Amplification and detection of difficult RNA targets

One Step RT-PCR Protocol Overview

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Detection of mRNAs 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.

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High thermal activity of the RT step

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 85°C, making this the best choice for detecting difficult RNAs, with high degrees of secondary structure.

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High 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.

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High reproducibility

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.

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Amplification and detection of difficult RNA targets

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 60°C. The four oligo pairs produced effective amplification bands despite the elevated GC content of the template RNA.

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ORDER INFORMATION

Each SunScript™ One Step RT-PCR Kit contains: Enzyme Mix, 2X Reaction Buffer and water. The complete manual is available under the Manual tab. Lucigen is an authorized distributor of Sygnis products in the US.

SunScript™ One Step RT-PCR Kit is intended for molecular biology use only and in vitro use only. This product is not intended for diagnosis, prevention or treatment of a disease in human beings or animals.