iScript™ gDNA Clear cDNA Synthesis Kit

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iScript™ gDNA Clear cDNA Synthesis Kit

Overview

Effective Removal of Genomic DNA during cDNA Synthesis

No amplification was observed from Bio-Rad iScript™ gDNA Clear cDNA Synthesis Kit (A), demonstrating complete removal of gDNA.

Effective Removal of Genomic DNA during cDNA Synthesis

1 µg of HeLa RNA was spiked in with 300 ng genomic DNA in two-step real-time PCR with (+RT) and without (-RT) reverse transcriptase. Both mRNA and gDNA were detected with the human TUBA assay.

Amplification was detected from other kits (B, QuantiTect Reverse Transcription Kit (QIAGEN); C, Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Fisher Scientific); D, PrimeScript RT Reagent Kit with gDNA Eraser (Takara), indicating a significant amount of residual gDNA.

RFU, relative fluorescence units

iScript™ gDNA Clear cDNA Synthesis Kit: Sensitivity and Reproducibility

Outstanding reproducibility over 7 orders of magnitude even after DNase treatment.

iScript gDNA Clear cDNA Synthesis Kit’s Sensitivity and Reproducibility

Tenfold serial diluted Jurkat RNA, ranging from 1 µg to 1 pg, was used to generate cDNA subsequently used for qPCR using the human B2M mRNA specific assay.

A, amplification profile; B, standard curve

Consistent Real-Time PCR Performance Observed Even after gDNA Removal

No changes in PCR efficiency, sensitivity, and reproducibility were observed.

Sample Efficiency R2 Slope
Treated (red dot) 100.3% 0.999 -3.314
Untreated (blue square) 99.0% 0.997 -3.345

Extracted Jurkat RNA was reverse transcribed using the iScript™ gDNA Clear cDNA Synthesis Kit with and without DNase treatment.

Two-step real-time PCR, using the human B2M mRNA specific assay, was then performed on samples with and without gDNA removal to monitor its impact on real-time PCR data.

Understanding Reverse Transcription
Which iScript™ Kit is right for me?
 

Most
Convenient

Integrated
DNA
Removal

Maximum
Input Use

Flexible
Priming
Options

Value
Solution

iScript Reverse Transcription
Supermix for RT-qPCR
iScript gDNA Clear
cDNA Synthesis Kit
iScript Advanced cDNA Synthesis Kit for RT-qPCR iScript Select cDNA
Synthesis Kit
iScript cDNA
Synthesis Kit
  • Gene expression
  • RNA quantification
  • Gene expression
  • RNA quantification
  • Gene expression
  • RNA quantification
  • Gene expression
  • Cloning
  • RNA quantification
  • Gene expression
  • RNA quantification
1 µg–1 pg 1 µg–1 pg 7.5 µg–100 fg 1 µg–1 pg 1 µg–100 fg
1 tube 3 tubes 2 tubes 5 tubes 2 tubes
  • 5x iScript RT Supermix
  • No-RT control supermix
  • 5x iScript RT Supermix
  • No-RT control supermix
  • DNase
  • DNase buffer
  • iScript Reverse Transcriptase
  • 5x iScript Advanced Reaction Mix
  • iScript Reverse Transcriptase
  • 5x iScript Reaction Mix
  • 3 priming options
  • iScript Reverse Transcriptase
  • 5x iScript Reaction Mix
26 min 36 min 21 min 40–90 min 26 min
High High High High High


The iScript gDNA Clear cDNA Synthesis Kit integrates an effective DNase digestion step with fast and sensitive first-strand cDNA synthesis for accurate qPCR data.

This kit combines the iScript Reverse Transcription Supermix and a specially formulated DNase to effectively eliminate genomic DNA (gDNA) from purified RNA in 10 minutes. qPCR-ready cDNA free of contaminant gDNA is generated in 36 minutes.

Key Features and Benefits

  • Effective genomic DNA removal — without compromising cDNA synthesis efficiency
  • Proprietary DNase buffer — maintaining RNA integrity during gDNA removal
  • Convenient all-in-one, single-tube supermix — for reproducible and efficient cDNA synthesis
  • Ready-to-use no-RT control supermix provided — for monitoring gDNA contamination
  • Superior sensitivity and reproducibility — works with a broad linear dynamic range of input total RNA (1 µg–1 pg)
  • Unbiased qPCR data — consistent efficiency, sensitivity, and reproducibility in qPCR results even after gDNA removal

gDNA-free cDNA ready in 36 minutes
This kit allows you to effectively remove contaminating gDNA and reliably synthesize cDNA with minimal hands-on time in an easy-to-follow protocol, resulting in cDNA free of genomic DNA in 36 minutes.

Workflow for DNA Removal and cDNA Synthesis

Importance of Addressing gDNA Contamination in RT-qPCR Gene Expression

gDNA contamination in RNA samples can be amplified in RT-qPCR, biasing the results for the transcript of interest. This problem is especially acute for low expressing genes — even a few copies of gDNA contamination can shift Cq values dramatically.

Benefit of Using Reverse Transcriptase with RNase H+ in RT-qPCR Gene Expression

RNase H works to degrade RNA that is hybridized to DNA. During cDNA synthesis, the RNase H domain of the Moloney Murine Leukemia Virus (MMLV) reverse transcriptase degrades the template RNA as the cDNA is synthesized.

This activity ensures the one-to-one conversion of RNA into cDNA molecules, resulting in more accurate gene expression data. Reverse transcriptase in all iScript reverse transcription reagents exhibits RNase H activity.

Choose the Right iScript Kit for Your Needs

Feature Integrated gDNA Removal Most Convenient Just Add RNA Maximum Input Use of up to 7.5 µg RNA Flexible Priming Options Value Solution
Product gDNA Clear cDNA Synthesis Kit Reverse Transcription Supermix Advanced cDNA Synthesis Kit Select cDNA Synthesis Kit cDNA Synthesis Kit
Total Input RNA 1 µg–1 pg 1 µg–1 pg 7.5 µg–100 fg 1 µg–1 pg 1 µg–100 fg
cDNA ready in 36 min 26 min 21 min 40–90 min 26 min

Related Products

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Generates reliable results, due to patented Sso7d fusion protein technology, in dye-based qPCR under various challenging conditions.

Performs efficiently in challenging singleplex and duplex qPCR reactions across a broad range of reaction conditions.

Produces consistent and reliable dye-based qPCR results.

Performs reliably and reproducibly with singleplex and duplex reactions.

More Information

Our Understanding Reverse Transcription Tutorial brings you product information, performance data, interactive tutorials, 3D animated videos, and more.

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Waste Electrical and Electronic Equipment ("WEEE Directive")

This product contains electrical or electronic material. Learn more about Bio-Rad's EU Recycle Program

iScript™ gDNA Clear cDNA Synthesis Kit, 25 x 20 µl reactions

1725034
25 x 20 µl reactions, 100 µl 5x supermix, contains reverse transcriptase, RNase inhibitor, dNTPs, primers, MgCl2, stabilizers; 200 µl no-RT control supermix; 12.5 µl DNase; 150 µl DNase buffer

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$210.00
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iScript™ gDNA Clear cDNA Synthesis Kit
iScript™ gDNA Clear cDNA Synthesis Kit, 100 x 20 µl reactions

1725035
100 x 20 µl reactions, 400 µl 5x supermix, contains reverse transcriptase, RNase inhibitor, dNTPs, primers, MgCl2, stabilizers; 400 µl no-RT control supermix; 50 µl DNase; 150 µl DNase buffer

List Price:
 
$520.00
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Number Description Options
6090 Amplification Reagents and Plastics Brochure, Rev E Click to download [ Add to Cart (Free) ]
6746 iScript™ Reverse Transcription Kits Flier, Ver B Click to download
6742 iScript™ gDNA Clear cDNA Synthesis Kit Product Information Sheet, Ver B Click to download
10043078 iScript™ gDNA Clear cDNA Synthesis Kit Product Insert, Ver B Click to download
6816 Protocol for Multiplex RT-qPCR Analysis Using PrimePCR™ Assays, Ver A Click to download