ReadiScript™ RT Reverse Transcription Kit

GAPDH gene expression analysis using TAQuest™ qPCR Master Mix with Helixyte™ Green *Low ROX*. Total RNA was extracted from HeLa cells, and cDNA was amplified in replicate reactions (1000, 100, 10, 1, 0 ng- From left to right) using the ReadiScript™ RT Reverse Transcription Kit. 2 µL volumes were used for each reaction for the qPCR detection of GAPDH.

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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22

Overview SDS Protocol

The ReadiScript™ RT Reverse Transcription kit is a convenient tool for cDNA synthesis used in gene expression analysis with qPCR. The two-tube kit format helps to set up the reaction faster and is optimized to yield cDNA over a broad dynamic range. The 5X supermix contains a unique blend of oligo(dT) and random primer mix to prevent the 5’ and 3’ bias of the target genes. The reaction mix contains a potent blend of RNase inhibitors to protect RNA during the setup and reverse transcription process. The resulting cDNA product is compatible with all qPCR chemistries or conventional end-point RT-PCR.

Platform

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Thermal Cycler

Components

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Allow the ReadiScript™ RT Reaction Mix (5X) to thaw on ice. Before using, vortex reaction mix thoroughly.

The following protocol can be used as a guideline.

Prepare the cDNA synthesis reaction by adding the following components to a microcentrifuge tube in the order they appear below:

Table 1. Reagents composition per well for each reaction.

Components Volume (20 µL/reaction)
ReadiScript™ RT Reaction Mix (5X) 5 µL
RNA 1-5 µL (Up to 1 µg)
RNase-Free Water Up to 20 µL
Carefully mix the reagents with a gentle vortex followed by a brief centrifuge.
Incubate the reaction in a thermal cycler at 65°C for 5 minutes.
Cool the reaction at room temperature to allow the primers to anneal to the DNA (approximately 5 minutes).
Add 1 µL of ReadiScript™ RT Enzyme to the reaction.
Carefully mix the reagents with a gentle vortex followed by a brief centrifuge.
Incubate the reaction in a thermal cycler at 25°C for 5 minutes.
Incubate the reaction in a thermal cycler at 42°C for 60 minutes.
Terminate the reaction by incubating the reaction at 65°C for 10 minutes.
Place the completed cDNA synthesis reaction on ice for use in downstream applications. For long-term storage, the reaction can be stored at -20 °C.

Images

Figure 1. GAPDH gene expression analysis using TAQuest™ qPCR Master Mix with Helixyte™ Green *Low ROX*. Total RNA was extracted from HeLa cells, and cDNA was amplified in replicate reactions (1000, 100, 10, 1, 0 ng- From left to right) using the ReadiScript™ RT Reverse Transcription Kit. 2 µL volumes were used for each reaction for the qPCR detection of GAPDH.

Documents

Safety data sheet
Product protocol
Certificate of analysis

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Components	Volume (20 µL/reaction)
ReadiScript™ RT Reaction Mix (5X)	5 µL
RNA	1-5 µL (Up to 1 µg)
RNase-Free Water	Up to 20 µL