Bio-Rad Laboratories

cDNA Synthesis

iScript cDNA Synthesis Kit

The iScript cDNA synthesis kit is a sensitive and easy-to-use system for first-strand cDNA synthesis. It contains the RNase H⁺ iScript reverse transcriptase for sensitivity, a premixed RNase inhibitor to prevent indiscriminate degradation of RNA template, and a unique blend of oligo(dT) and random primers. All components are optimized to yield sensitive, unbiased representation over a broad dynamic range, with minimal setup and reaction time. With the iScript cDNA synthesis kit, great results have never been easier.

Comparison of Reaction Assemblies and Protocols
Bio-Rad Laboratories' iScript cDNA Synthesis Kit	Competitor I's First-Strand cDNA Synthesis System
To a 0.2 ml tube, add:	To a 0.2 ml tube, add:
5X cDNA synthesis kit buffer iScript enzyme mixture Nuclease-free water RNA sample	Oligo(dT) primers Random hexamers Buffer MgCl₂ dNTPs DTT RNase inhibitor Reverse transcriptase Nuclease-free water RNA sample
Next, program the thermal cycler to run the following protocol:
5 min at 25°C 30 min at 42°C 5 min at 85°C Hold at 4°C	5 min at 65°C Remove, quick chill on ice Add additional items Mix and incubate 2 min at 42°C Add reverse transcriptase Return reaction to thermal cycler 50 min at 42°C 15 min at 70°C Hold at 4°C RNase H digestion, 20 min at 37°C
Total time, including setup and reaction:
45 min	>110 min

The iScript cDNA synthesis kit represents a major simplification in reverse transcription. Reaction optimization and premixing have already been done in the iScript cDNA synthesis kit. With only three optimized solutions to add to your RNA sample, reaction assembly occurs in just one step. Once assembled, you can just program the thermal cycling protocol and move on to something else. Conversely, competitor I's kit is tedious and requires you to remain at the laboratory bench during assembly and the course of the reaction. Competitor I's kit requires multiple incubations and additions of reaction components, increasing both the time to completion and the risk of contamination.

Comparison of amplification success using iScript and competitor I's reverse transcriptase. Input RNA from 1 µg to 1 pg was reverse transcribed, amplified using iQ SYBR Green supermix, and detected on the iCycler iQ real-time PCR detection system. Top, Bio-Rad iScript cDNA synthesis kit delivers at least 6 orders of dynamic range while maintaining optimum sensitivity with limited amounts of input RNA. Bottom, competitor I's reverse transcriptase fails to deliver resolution over a wide dynamic range or sensitivity with a limited amount of input RNA.

Efficiency and performance of iScript across a broad range of concentrations in quantitative RT-PCR. Quantitative PCR was performed on serial dilutions of cDNA (red) or on cDNAs (blue) from serial dilutions of input total RNA. The resulting standard curves are linear and virtually superimposable.

Sensitivity of the iScript cDNA synthesis kit in RT-PCR. cDNA was synthesized at 42°C amplified with iTaq DNA polymerase using beta-actin primers. Lane M, EZ Load 100 bp molecular ruler; C, no-template negative control.