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One-Step Quantitative RT-PCR

The iScript one-step quantitative RT-PCR kits are highly sensitive solutions for real-time quantitative RT-PCR on a broad range of optical thermal cyclers. cDNA synthesis and PCR amplification are conveniently carried out in the same tube, using the powerful iScript RNase H+ reverse transcriptase and iTaq hot-start DNA polymerase combination.

iScript One-Step RT-PCR Kit With SYBR Green

The iScript one-step RT-PCR kit with SYBR Green is optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity. The proprietary reaction buffer has been specifically formulated for optimal activity of both iScript reverse transcriptase and iTaq DNA polymerase, while minimizing the potential for primer-dimer formation and other nonspecific PCR artifacts. Thus, with this kit, clean detection of low-copy targets is easy to achieve. The kit is perfectly suited for a broad range of real-time PCR applications.

  • Highly specific amplification over a broad dynamic range
  • Extremely sensitive detection down to 100 fg of input RNA
  • Convenient one-tube reaction setup minimizes handling and contamination risk

The iScript one-step RT-PCR Kit with SYBR Green provides high sensitivity across a broad range of concentrations. One-Step RT-PCR reactions were performed in triplicate, along with no-template controls, using GAPDH primers and 100 ng to 100 fg of total HeLa RNA. Reactions were carried out on the iCycler iQ real-time system. Standard curve had r = 1.000, slope = –3.466, efficiency = 95%.

Melt-curve analysis of RT-PCR reactions performed using the iScript one-step RT-PCR kit with SYBR Green shows absence of nonspecific PCR artifacts. Melt-curve protocol was performed subsequent to the RT-PCR shown above, revealing a single clean melting peak for all samples tested.

iScript One-Step RT-PCR Kit for Probes

The iScript one-step RT-PCR kit for probes is designed for use with all types of hybridization probes, including dual-labeled oligonucleotide probes, FRET probes, and molecular beacons.

The kit is designed to promote specific amplification over a broad dynamic range, while preventing the formation of primer-dimers and other nonspecific PCR artifacts. The magnesium concentration has been stringently optimized to support probe hybridization and hydrolysis, as well as duplex RT-PCR reactions.

  • Sensitive and specific reverse transcription using any hybridization probe chemistry
  • Optimized reaction mixture minimizes the need for reaction optimization
  • Convenient one-tube reaction setup minimizes handling and contamination risk
  • Unique capability to perform duplex reactions

The iScript one-step RT-PCR kit for probes delivers unparalleled results over an extremely wide dynamic range. RNA (1 µg to 100 fg) isolated from HeLa cells using the Aurum total RNA kit was reverse-transcribed and amplified using primers to beta-actin and a FAM-labeled detection probe. Each dilution was performed in triplicate, and RT-PCR was carried out on the iCycler iQ real-time detection system. Standard curve had r = 1.000, slope = –3.39, and efficiency = 97.2%.