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Working with RNA samples
Because real-time PCR (qPCR) cannot be performed directly on RNA, RNA samples must first be copied to complementary DNA (cDNA) in a process called reverse transcription (RT). After reverse transcription, the cDNA can then be amplified using qPCR.
Applications that start with RNA samples include gene expression analysis detecting messenger RNA (mRNA) and virus detection looking at viral RNA. Both applications offer either a one-step or two-step qPCR workflow, each with its own benefits and drawbacks.
DNA samples do not require the reverse transcription step and can be directly amplified by qPCR.
One-step vs two-step workflow for RNA targets
One-step workflow
A one-step master mix enables both reverse transcription and qPCR to be performed in one tube. It includes everything required to reverse transcribe RNA to cDNA and then amplify DNA by qPCR. There is no need to open the tube to add additional reagents.
The one-step reaction has fewer steps, less pipetting, and a lower risk of cross-contamination. It can be automated using robotics and thus allows for high-throughput sample processing. One-step is ideal for testing many samples with one to a few target sequences. One drawback is that the cDNA cannot be stored since it is part of the one-step reaction. So, if you need to use your cDNA to analyze many targets, use a two-step workflow.
Two-step workflow
A two-step workflow involves performing reverse transcription first, to synthesize cDNA. Once completed, a small portion of the cDNA is transferred to a separate tube along with qPCR master mix and a target-specific assay. qPCR amplification of cDNA is then performed.
The cDNA generated during the reverse transcription step can be stored for further testing. Two-step is the better choice when customers need to screen many target sequences in each sample. It does, however, involve more steps and more pipetting.
Workflow comparison
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One-step workflow |
Two-step workflow |
Features |
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Use case |
Use when testing many samples with one or few targets |
Use when testing fewer samples with many targets |
Primary consideration |
cDNA cannot be stored |
Multiple steps, slower to obtain results |
Featured one-step and two-step master mixes
One-step master mixes |
Description |
Designed for fast, highly-sensitive real-time RT-PCR, even in the presence of challenging PCR inhibitors |
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High concentration (4X) master mix designed for robust and reproducible one-step pathogen detection and gene expression workflows. |
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General purpose reagent provides sensitive, reproducible detection of up to four different targets in a single multiplex reaction. Available in No ROX and Mustang Purple versions. |
Two-step master mixes |
Description |
Provides best-in-class performance, including the widest quantifiable range, in both single and duplex reactions, even with challenging targets. |
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High-performing, high-throughput master mixes for genotyping and copy number variation (CNV). Delivers exceptional specificity, dynamic range, and reproducibility even in the presence of inhibitors. |
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A 2X formulation designed for gene expression and miRNA analysis, containing a thermostable fast DNA polymerase and dNTPs in an optimized buffer solution for maximum robustness and reproducibility. |
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A 2X master mix optimized for rapid low-level microbial detection even in the presence of PCR inhibitors. Available in two formulations: ROX to address up to 3 targets, and no ROX for high-order multiplexing of more than 3 targets. |
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Designed to reduce pipetting errors via tracking dye. Formulated for excellent specificity, single-copy detection, and wide instrument compatibility. |
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Provides exceptional specificity with dual hot-start mechanism. Formulated for exceptional specificity, single-copy detection, and wide instrument compatibility. |
*TaqPath master mixes are general purpose reagents labeled 'For Laboratory Use' and manufactured in an ISO 13485-certified and FDA-registered facility that adheres to cGMP principles.
For Research Use Only. Not for use in diagnostic procedures.