iScript™ Reverse Transcription Supermix for RT-qPCR

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iScript Reverse Transcription Supermix for RT-qPCR

Overview

Broad Linear Dynamic Range

The iScript™ reverse transcription supermix for RT-qPCR efficiently reverse transcribes RNA over a broad linear dynamic range for reliable gene expression analysis data. Different amounts of HeLa cell RNA (amounts shown in insert) were reverse transcribed and one-tenth of resulting cDNA was used as template to amplify β-actin gene (~90 bp) in 20 µl qPCR reactions with iQ™ SYBR® Green supermix. Standard curve r2 = 0.999, efficiency = 99.7%, slope = –3.33. RFU, relative fluorescence units.

Data Reproducibility

Excellent data reproducibility. PGK-1 mRNA (~160 bp), a gene that encodes a glycolytic enzyme, was quantified using iScript™ reverse transcription supermix for RT-qPCR both with 100 ng () and 100 pg () of input RNA. For each input RNA, 48 individual RT reactions were performed and one-tenth of resulting cDNA was used in the qPCR reaction with SsoFast™ probes supermix. The gene expression analysis data show excellent reproducibility both with high and low levels of input target mRNA. The ~10 threshold cycle (CT) difference for the 1000-fold dilution of RNA (100 ng–100 pg) demonstrates good reverse transcription efficiencies across different input RNAs. RFU, relative fluorescence units.

Unbiased Representation of 5' and 3' Regions

Unbiased representation of 5' and 3' regions of target genes. Reverse transcription of 100, 10, 1, and 0.1 ng input RNA was performed with iScript™ reverse transcription supermix for RT-qPCR. Primer pairs were designed at 5' (, ~60 bp) and 3' (, ~70 bp) ends of the MAP gene and qPCR was performed with one-tenth of input cDNA using iQ™ SYBR® Green supermix. There were no significant differences (<0.5 threshold cycle [CT] difference) between the two primer pairs, which demonstrates unbiased representation of 5' and 3' regions. RFU, relative fluorescence units.

Protection of RNA Integrity

Potent RNaseA inhibition. iScript™ reagents for RT-qPCR include an optimum blend of RNaseA inhibitor for protecting RNA integrity. Reverse transcription was performed using 0.1 pg of input RNA with iScript reagent alone (), spiked with RNaseA (), or spiked with RNaseA without the RNaseA inhibitor included in the reaction (). 18S rRNA (~70 bp) was amplified using iQ™ SYBR® Green supermix. A significant threshold cycle (CT) delay was observed when the reaction included RNaseA but no RNaseA inhibitor, which demonstrates potent RNaseA inhibition. RFU, relative fluorescence units.

Maximum Efficiency and Sensitivity with SYBR® Green

SYBR® Green compatibility with iScript™ reverse transcription supermix for RT-qPCR. Different amounts of HeLa cell RNA were reverse transcribed and one-tenth of resulting cDNA was used as template to amplify 18S rRNA (~70 bp), β-actin (~90 bp), GAPDH (~200 bp), and MAP (5' amplicon: ~60 bp; 3' amplicon: ~70 bp) genes in 20 µl qPCR reactions with iQ™ SYBR® Green supermix. CT, threshold cycle.

Maximum Efficiency and Sensitivity with EvaGreen

EvaGreen and high-GC amplicon compatibility with iScript™ reverse transcription supermix for RT-qPCR. Different amounts of HeLa cell RNA were reverse transcribed and one-tenth of resulting cDNA was used as template to amplify CBP (~100 bp) and β-actin (~90 bp) genes in 20 µl qPCR reactions with SsoFast™ EvaGreen® supermix. CT, threshold cycle.

Maximum Efficiency and Sensitivity with SsoFast™ Probes Supermix

Probe-based qPCR with SsoFast probes supermix. Different amounts of HeLa cell RNA were reverse transcribed and one-tenth of resulting cDNA was used as template to amplify β-actin (~90 bp) and α-tubulin (~120 bp) genes in 20 µl qPCR reactions. CT, threshold cycle.

Maximum Efficiency and Sensitivity with iQ™ Supermix

 

Probe-based qPCR with iQ supermix. Different amounts of HeLa cell RNA were reverse transcribed and one-tenth of resulting cDNA was used as template to amplify β-actin (~90 bp) and α-tubulin (~120 bp) genes in 20 µl qPCR reactions. CT, threshold cycle.

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The iScript reverse transcription supermix for RT-qPCR is a simple, fast, and sensitive first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR. In one tube, the preblended 5x supermix contains all the necessary components except RNA template for reverse transcription.

  • Increase reproducibility of reverse transcription and reduce errors during setup — 1-tube format for simple and fast setup and reduced pipetting variability
  • Detect low-level target genes and conserve RNA during gene expression analysis — broad linear dynamic range of total input RNA (1 µg–1 pg) with a highly efficient RNase H+ MMLV reverse transcriptase
  • Use with dilute RNA samples — 5x concentration of supermix allows for greater volume of RNA sample in a 20 µl cDNA synthesis reaction
  • Eliminate freeze/thaw cycles — liquid format at –20ºC shortens overall processing time and maximizes stability
  • Validate purity of input RNA and gene expression results — optional no-RT controls for detecting genomic DNA contamination
  • Obtain accurate results — potent blend of RNase inhibitor prevents RNA degradation during reaction setup and reverse transcription
  • Increase primer design flexibility and prevent 5' and 3' bias — optimum blend of oligo(dT) and random primers in the 5x supermix for complete RNA coverage
  • Complete cDNA synthesis and qPCR the same day — fast 26 minute cDNA synthesis protocol
Storage at –20ºC Guaranteed for 12 months in a constant temperature freezer (this reagent will not freeze at –20ºC; multiple freeze thaws not recommended)
Number Description Options
10020178 iScript™ Reverse Transcription Supermix for RT-qPCR Product Insert, Ver C Click to download
6031 iScript™ Reverse Transcription Supermix for RT-qPCR Product Information Sheet, Rev B Click to download
6746 iScript Reverse Transcription Kits Flier Click to download [ Add to Cart (Free) ]
6252 Reagent Comparison Guide for Real-Time PCR Brochure, Rev C Click to download [ Add to Cart (Free) ]
6090 Amplification Reagents and Plastics Brochure Click to download [ Add to Cart (Free) ]
6763 Gene Expression Real-Time PCR Workflow Flier Click to download

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