1-Step Quantitative Reverse Transcription PCR (RT-qPCR) from RNA

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1-Step Quantitative Reverse Transcription PCR (RT-qPCR) from RNA

Overview

Superior Dynamic Range and Sensitivity Performance of iTaq Universal SYBR® Green One-Step Kit

A cell lysate and a 226 bp human GAPDH assay were used to determine dynamic range and sensitivity performance of different kits on a CFX384 real-time PCR instrument.

A. The iTaq Universal SYBR® Green One-Step Kit

B. The Qiagen QuantiTect SYBR® Green FT-PCR kit

C. Life Technologies Power SYBR RNA-to-CT kit

iTaq Universal SYBR® Green One-Step Kit performed better than the other kits tested in terms of cell input, PCR efficiency and R2


  iTaq Universal SYBR® Green One-Step Kit (Bio-Rad) QuantiTect SYBRGreen RT-PCR Kit (Qiagen) Power SYBRRNA-to-Ct Kit (Life Technologies)
Cell Input 1,000 to 0.001 1,000 to 1 100 to 0.1
PCR Efficiency 96.8% 94.5% 83.3%
R2 0.997 0.977 0.981
Greater Sensitivity with iTaq Universal Probes One-Step Kit

Bio-Rad’s iTaq Universal Probes One-Step Kit (A, C) and TaqMan RNA-to-CT 1-step kit from Life Technologies (B, D) were used in high-performing duplex and singleplex RT-qPCR reactions. Input RNA was 10–100 ng. Reactions were performed on a CFX96 real-time PCR detection system.

The iTaq Kit exhibits a slight 2–quantification cycle (Cq) delay from singleplex to duplex, which is typical for duplex reactions; however, the TaqMan kit exhibits a 4-Cq delay (greater than one log of dynamic range), which indicates less sensitivity.

Duplex PGK1, PPIA Assay

iTaq universal probes one-step kit
iTaq Universal Probes One-Step Kit
TaqMan RNA-to-CT 1-step kit (Life Technologies)
TaqMan RNA-to-CT 1-step kit (Life Technologies)

Singleplex PGK1 Assay

iTaq universal probes one-step kit
iTaq Universal Probes One-Step Kit
TaqMan RNA-to-CT 1-step kit (Life Technologies)
TaqMan RNA-to-CT 1-step kit (Life Technologies)
Superior Dynamic Range, Sensitivity, and Linearity with GC-Rich Template

Superior dynamic range and sensitivity performance of iTaq Universal SYBR® Green One-Step Kit.

A 93-bp humanBAX assay was performed with 500 ng to 5 pg of total RNA on an AB ViiA 7 real-time PCR instrument.

The delayed cycle threshold (Ct) values, loss of dynamic range and the 500 ng input, and compression of the 50 ng input in the Power SYBR® RNA-to-Ct kit demonstrated RT-qPCR inhibition at higher RNA inputs and lack of low-level target expression detection. ΔRn, baseline-corrected normalized reporter.

iTaq Universal SYBR<sup>®</sup> Green one-step kit PCR efficiency of 102.4% and R2 of 0.999 Life Technologies Power SYBR<sup></sup> RNA-to-Ct kit PCR efficiency of 111.8% and R2 of 0.992
iTaq Universal SYBR® Green One-Step Kit PCR efficiency of 102.4% and R2 of 0.999 Life Technologies Power SYBR® RNA-to-Ct kit PCR efficiency of 111.8% and R2 of 0.992

Optimal RT-qPCR efficiency and sensitivity with iTaq Universal Probes One-Step Kit.

A 72% GC-rich CBP assay with an input of 100 ng–10 pg RNA was performed on a CFX96 real-time PCR detection system.

Brilliant III ultra-fast QRT-PCR master mix (Agilent Technologies) produced quantification cycle (Cq) values with less than optimal data precision for the technical replicates.

iTaq Universal SYBR<sup>®</sup> Green one-step kit PCR efficiency of 98.1% and R2 of 0.999 Brilliant III ultra-fast QRT-PCR master mix (Agilent Technologies) PCR efficiency of 87.3% and R2 ≥0.984
iTaq Universal SYBR® Green One-Step Kit PCR efficiency of 98.1% and R2 of 0.999 Brilliant III ultra-fast QRT-PCR master mix (Agilent Technologies) PCR efficiency of 87.3% and R2 ≥0.984

 

Inhibitor Tolerance and Multiplexing

Superior RT-qPCR performance in a triplex reaction with iTaq Universal Probes One-Step Kit.

An XPO (FAM), TUB (HEX), and GAPDH (Quasar) assay set with an input of 100 ng–100 fg RNA was performed on a CFX96 Real-Time PCR Detection System using the iTaq Kit and two competitor products.

 
Dye Logs Standard Curve, R2 Efficiency, %
 FAM 5 0.998 106
 HEX 4 0.999 101
 Quasar 6 0.99 103

The iTaq Universal Probes One-Step Kit (A) exhibited the greatest dynamic range across all three assays, which indicates a high degree of sensitivity. In addition, the iTaq Universal Probes One-Step Kit demonstrated the lowest standard deviations for technical replicates.

Dye Logs Standard Curve, R2 Efficiency, %
 FAM 4 0.998 101
 HEX 4 0.993 97.6
 Quasar 4 0.998 97.5

Brilliant III ultra-fast QRT-PCR master mix from Agilent Technologies (B) and TaqMan RNA-to-CT 1-step kit from Life Technologies (C) did not perform as well, exhibiting four logs of dynamic range for each assay (lower sensitivity) and higher standard deviations for technical replicates (lower data precision).

Dye Logs Standard Curve, R2 Efficiency, %
 FAM 4 0.999 96.7
 HEX 4 0.999 96.7
 Quasar 4 0.999 95.6
 

Great ethanol tolerance of iTaq Universal Probes One-Step Kit

Final concentration 10 pg 200 pg
Average Cq dCq Average Cq dCq
0% 27.4 - 23.0 -
0.25% 27.3 -0.1 22.9 -0.1
1.25% 27.8 0.4 23.4 0.4

Singleplex reactions were prepared using iTaq Universal Probes One-Step Kit with two RNA inputs of 10 pg and 200 pg and ethanol was spiked in at a final concentration of 0%, 0.25, and 1.25% to evaluation the tolerance of the kit. The iTaq Universal Probes One-Step Kit resulted in less than 0.5 dCq between reactions indicating minimal inhibition by ethanol.


Superior RT-qPCR inhibitor tolerance of iTaq Universal SYBR® Green One-Step Kit.

A human ACTB assay was performed with 200 ng of total RNA, and chocolate and heparin were spiked in to evaluate the tolerance of the iTaq Universal SYBR® Green One-Step Kit () versus the Life Technologies Power SYBR RNA-to-Ct () and Qiagen QuantiTect SYBR® Green RT-PCR kit ().        

A, Chocolate spiked in from 0.01 to 0.2% resulted in a greater quantification cycle (Cq) delay from the control sample with the Life Technologies Power SYBR RNA-to-Ct kit (), indicating less tolerance for RT-qPCR inhibitors than the iTaq Universal SYBR® Green One-Step Kit ().

B. Heparin spiked in from 200 to 3,200 pg/µl caused the 17.83 Cq delay at 200 pg/µl and eventual complete inhibition with the Life Technologies Power SYBR RNA-to-Ct kit () but only minor Cq delays with the iTaq Universal SYBR® Green One-Step Kit ()

C. Heparin spiked in from 200 to 1,600 pg/µl produced a greater Cq delay from the control (unspiked) sample with the Qiagen QuantiTect SYBR® Green RT-PCR kit () than the iTaq Universal SYBR® Green One-Step Kit ().

Superior Data Precision and Reproducibility

iTaq Universal Probes One-Step Kit

Superior data precision and reproducibility with iTaq universal probes one-step kit

GAPDH assay with input of 8.78 to 100 ng of RNA shows high precision and reproducibility of results. PCR was performed on a CFX96 Real-Time Detection System (RFU, relative fluorescence units.)

iTaq Universal SYBR Green One-Step Kit: Superior precision compared to other kits.

A human TUBA assay with an input of 8.78 to 100 ng of total RNA was performed on an ABI 7900 real-time instrument using different RT-PCR kits (ΔRn, baseline-corrected normalized reporter.)

The iTaq universal SYBR<sup>®</sup> Green one-step kit demonstrated robust data acquisition even with small-fold changes.

A. The iTaq Universal SYBR® Green One-Step Kit

 
The Qiagen QuantiTect SYBR<sup>®</sup> Green RT-PCR kit and Life Technologies Power SYBR precision RNA-to-Ct kit

B. The Qiagen QuantiTect SYBR® Green RT-PCR kit

 
Both showed less than optimal precision. ΔRn, baseline-corrected normalized reporter.

C. Life Technologies' Power SYBR® precision RNA-to-Ct kit

iTaq Universal One-Step RT-qPCR Kits provide high PCR efficiency in sensitive SYBR<sup>&reg;</sup> Green or probe assays, even with difficult targets and challenging samples. Ideal for high-throughput RT-qPCR screening.


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Ordering Information


Description

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iTaq Universal One-Step RT-qPCR Kits are a fast and convenient solution for real-time PCR using the powerful combination of iScript RNase H+ MMLV reverse transcriptase and hot-start iTaq DNA Polymerase in one reaction, providing improved PCR efficiency, wider dynamic range, superior sensitivity and specificity, and inhibitor tolerance without affecting performance.

Available for SYBR®Green and probe-based qPCR assays, iTaq Universal One-Step Kits are ideal for low-to mid-throughput real-time PCR screening and validation. A simplified work flow and reduced cycling times enable screening and validation of the greatest number of samples and targets in the shortest period of time.

Features and Benefits of iTaq Universal One-Step RT-qPCR Kits

  • Enhanced efficiency, specificity, and sensitivity— our patented inhibitor reducer* prevents interference of the DNA polymerase by the RT enzyme during cDNA replication
  • Compatible with all real-time PCR instruments — our universal reference dye enables ROX normalization of qPCR data regardless of the ROX level requirements of the qPCR system
  • Superior gene expression results under various cycling conditions — our robust formulation delivers consistent performance in standard or fast cycling conditions, primer concentrations, sample and target types, and temperature ranges, allowing cost and time savings
  • Obtain superior results with singleplex and multiplex reactions — advanced formulation enables the simultaneous amplification of up to two targets, resulting in higher data precision with fewer pipet steps and reduced sample usage

Instrument Compatibility

iTaq Universal One-Step RT-qPCR Kits are compatible with Bio-Rad Real-Time PCR Detection Systems and commercially available systems listed below:

Systems
Applied Biosystems
  • StepOne/StepOnePlus
  • 7500, ViiA 7
  • 7000, 7300, 7700, 7900HT, QuantStudio Systems
Stratagene
  • Mx3000P, 3005P, 4000
Eppendorf
  • Mastercycler ep realplex 2, 4
QIAGEN
  • Corbett Rotor-Gene 3000, 6000, Q
Roche
  • LightCycler 480, 96
  • LightCycler 1.0, 1.5, 2.0*
BioFire
  • LightScanner HR-1
  • LightScanner 32*

* BSA must be added according to instrument specifications

Applications and Uses of iTaq Universal One-Step RT-qPCR Kits

  • Gene expression analysis
  • Multiplexing
  • Absolute quantification
  • Mutation detection
  • Pathogen detection
  • Viral detection
  • Genetically modified organisms (GMO) characterization
  • Genetic profiling

Related Categories

Supporting Documentation

Online Resources

* U.S. patent 8,338,094.


Specifications

Product format Two-tube system
Kit content iScript reverse transcriptase + 2x iTaq PCR reaction mix
Detection chemistry SYBR Green or probes
Multiplex capability (for probes only) Duplex (triplex possible depending on the abundance of the targets)
Inhibitor tolerance Tested with ethanol. Also compatible with up to 4 µl of crude lysates
5’ to 3’ exonuclease activity of the enzyme(s) Yes
Hot-start, activation time Yes, 1–3 min
Sample input 100 fg to 100 ng
Sensitivity 10 copies in a duplex reaction
Reference dye Patented universal dye preblended in the iTaq reaction mix
Instrument compatibility Both ROX-dependent (high- and low-) and -independent instruments
Product expiration (after shipping date) 12 months
RT storage at –20°C (after shipping date) 12 months
Reaction mix storage at 4°C (after thawing) Up to 3 months
Customer guarantee (minimum usage time) at –20°C 12 months

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iTaq™ Universal SYBR® Green One-Step Kit, 100 x 20 µl rxns, 1 ml

1725150
100 x 20 µl reactions, 1 ml (1 x 1 ml), one-step RT-PCR kit, includes 2x one-step SYBR® Green reaction mix containing iTaq DNA polymerase, buffers, and ROX normalization dyes; iScript™ reverse transcriptase; and nuclease-free water

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iTaq™ Universal SYBR® Green One-Step Kit, 500 x 20 µl rxns, 5 ml (5 x 1 ml)

1725151
500 x 20 µl reactions, 5 ml (5 x 1 ml), one-step RT-PCR kit, includes 2x one-step SYBR® Green reaction mix containing iTaq DNA polymerase, buffers, and ROX normalization dyes; iScript™ reverse transcriptase; and nuclease-free water

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iTaq Universal Probes One-Step Kit, 100 x 20 µl rxns, 1 ml

1725140
100 x 20 µl reactions, 1 ml (1 x 1 ml) 2x one-step reaction mix, containing iTaq DNA polymerase, buffers, and ROX normalization dyes; iScript reverse transcriptase; and nuclease-free water

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$236.00 
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iTaq Universal Probes One-Step Kit, 500 x 20 µl rxns, 5 ml (5 x 1 ml)

1725141
500 x 20 µl reactions, 5 ml (5 x 1 ml) 2x one-step reaction mix containing iTaq DNA polymerase, buffers, and ROX normalization dyes; iScript reverse transcriptase; and nuclease-free water

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$861.00 
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iTaq Universal Probes One-Step Kit, 5,000 x 20 µl rxns

12013250
5,000 x 20 µl reactions, 50 ml (1 x 50 ml) 2x one-step reaction mix containing iTaq DNA polymerase, buffers, and ROX normalization dyes; 2.5 ml (1 x 2.5 ml) iScript reverse transcriptase; and 45 ml (1 x 45 ml) nuclease-free water

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Documents

Number Description Options
10032048 iTaq™ Universal SYBR® Green One-Step Kit Product Insert, Rev B Click to download
10032046 iTaq Universal Probes One-Step Kit Product Insert, Rev B Click to download
6252 Reagent Comparison Guide for Real-Time PCR Brochure, Rev C Click to download [ Add to Cart (Free) ]
7178 Amplification: Real-Time PCR Reagents and Kits Brochure Click to download
6090 Amplification Reagents and Plastics Brochure Click to download [ Add to Cart (Free) ]
6763 Gene Expression Real-Time PCR Workflow Flier Click to download
6290 PrimePCR Assays and Panels Brochure, Rev D Click to download
6516 Better, Faster, and Less Expensive Gene Expression Analysis Using Multiplex One-Step RT-qPCR: A Case Study of Colorectal Cancer, Rev A Click to download

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