cDNA Synthesis for qPCR

Reverse transcription combined with quantitative PCR, or RT-qPCR, is one of the most common RNA analysis methods. It allows detection and quantification of RNA for gene expression analysis, pathogen detection, disease research, and other applications. In the RT-qPCR workflow, two common approaches can be used: one-step or two-step RT-qPCR.

In a two-step RT-qPCR workflow, RNA quantification is performed in two separate reactions. First, sample RNA is converted to complementary DNA, or cDNA, in the reverse transcription step; the first cDNA strand generated is then amplified and quantified by qPCR. One of the key advantages of the two-step process is flexibility with reaction setup and the possibility to analyze multiple targets from an RNA sample.

Learn more about RT-PCR

Since the accuracy of gene expression quantification largely depends on cDNA quality and quantity, the reverse transcription step is critical for successful RT-qPCR. Invitrogen SuperScript IV VILO Master Mix is a first-strand cDNA synthesis reaction mix designed specifically for two-step RT-qPCR. The master mix contains the proven Invitrogen SuperScript IV Reverse Transcriptase, leverages the VILO (Variable Input, Linear Output) technology, and has optimized buffer conditions for exceptional performance and linearity across the broadest range of input RNA.

Additionally, the SuperScript IV VILO Master Mix provides 3 levels of control that enables data accuracy and reproducibility in RT-qPCR reactions:

• SuperScript IV VILO Master Mix includes all the reaction components in a pre-mixed formulation. Just add the RNA template and nuclease-free water to complete the reaction mix.
• SuperScript IV VILO Master Mix No-RT control included in the kit, allows for monitoring of gDNA contamination and to avoid misleading results.
• SuperScript IV VILO Master Mix with ezDNase Enzyme removes contaminating gDNA while preserving RNA quality and RT-qPCR data accuracy.

SuperScript IV VILO Master Mix is known for its reliability, efficiency, robustness, ease of gDNA removal, and workflow simplicity. Click on the attributes below to see supporting data.

Super reliable Linearity across ten orders of magnitude for input RNA

Super-efficient Four times more cDNA, Ct values lower by two cycles

Super robust High cDNA yields with degraded or inhibitor-containing RNA samples

Super safe Fast, easy, and RNA-friendly gDNA removal

Super simple Time-saving, simplified workflow

SuperScript IV VILO Master Mix contains a proprietary helper protein which improves the interaction between SuperScript IV Reverse Transcriptase and the template RNA, and thereby, extends linearity across ten orders of magnitude for input RNA (Figure 1). Continue to get a high degree of linearity across a wide range of target inputs with our qPCR master mixes that are specifically formulated for highly sensitive and accurate gene expression analysis.

Figure 1. Linearity across 10 orders of magnitude for a range of RNA input. Serial dilutions of total RNA from HeLa cells were reverse transcribed using the SuperScript IV VILO Master Mix, followed by qPCR reactions using human TaqMan assay for 18S rRNA with the Invitrogen EXPRESS qPCR SuperMix Universal. Even across a wide range of RNA input from 1 fg to 1 μg, the master mix exhibits a coefficient of correlation of 0.999 and high efficiency of 94.2%. The amplification plot illustrates the robust nature of the SuperScript IV VILO Master Mix over a broad range of RNA input. This means that you can normalize your lower-abundance genes to your reference genes without worrying about potential variation of RT efficiency at different RNA input levels.

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The cDNA, generated from low RNA input, showed that the SuperScript IV VILO Master Mix had the highest efficiency, delivered greater cDNA yields, and lower Ct values when compared to eight other commercially available RNA to cDNA kits (Figure 2). With SuperScript IV VILO Master Mix, the average yield of cDNA was four times greater, and the Ct values were on average by two cycles earlier. The benefits of higher cDNA synthesis efficiency include:

• Possibility to use lower RNA inputs in your RT-qPCR workflow
• Better RT-qPCR sensitivity to work low-copy targets and degraded RNA
• Possibility to archive cDNA for future studies

Figure 2. Highest efficiency across a broad range of targets. cDNA synthesis was performed with different master mixes per manufacturer instructions, using 1 ng of total HeLa RNA input. qPCR was performed with Invitrogen EXPRESS qPCR SuperMix and Applied Biosystems TaqMan primer and/or probes for indicated gene targets. Delta C_t values (∆C_t= C_t – C_{tSuperScript IV VILO}) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield and on average two cycles lower C_t values compared to other RNA to cDNA synthesis kits.

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Often cDNA synthesis for qPCR requires high quality intact RNA samples to achieve accurate RT-qPCR results. However, SuperScript IV VILO Master Mix shows robust performance even when challenged with difficult samples, containing common reaction inhibitors (Figure 3A) or degraded RNA (Figure 3B).

Figure 3A. Exceptional performance with inhibitor-containing RNA. cDNA synthesis was performed using 100 ng HeLa in reactions containing different inhibitors. qPCR was performed with TaqMan primer/probes for the B2M gene target using EXPRESS qPCR SuperMix. Delta C_t values (∆C_t= C_t – C_{tSuperScript IV VILO}) show that SuperScript IV VILO Master Mix delivered maximum cDNA yield and minimal C_t values in presence of all tested reaction inhibitors.

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RNA purification methods are not foolproof and often fail to completely remove gDNA. Amplification of contaminating gDNA can cause a shift in Ct values, especially when detecting poorly expressed genes. DNase I enzyme is commonly used to remove gDNA from RNA. However, DNase I may degrade single stranded DNA such as primers and cDNA, and therefore DNase I must be inactivated or removed prior to cDNA synthesis. DNase I removal processes using EDTA or other methods can damage or reduce yields of RNA (Figure 4A).

The gDNA removal step is simplified with the SuperScript IV VILO Master Mix format with ezDNase enzyme. The Invitrogen ezDNase enzyme exhibits double stranded DNA-specific activity and allows gDNA removal in two minutes at 37°C. The ezDNase enzyme is thermolabile, and is inactivated at 50°C, which is the standard SuperScript IV RT cDNA synthesis temperature. This attribute of ezDNase eliminates the need for a separate inactivation step and enables accuracy and confidence in RT-qPCR results (Figure 4A and 4B).

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Due to the high processivity of SuperScript IV RT in the SuperScript IV VILO Master Mix, cDNA synthesis reactions can be significantly faster (10 minutes) in comparison to reactions performed with traditional RT enzymes (60 minutes). Furthermore, the protocol for gDNA removal with ezDNase takes as little as two minutes and does not require a dedicated enzyme inactivation step. Therefore, the workflow for cDNA synthesis involving gDNA removal with the SuperScript IV VILO Master Mix can be significantly shorter than with traditional RNA to cDNA kits (Figure 5).

Figure 5. Workflow comparisons. Comparison between the SuperScript IV VILO Master Mix cDNA synthesis workflow including sample treatment with ezDNase enzyme (top) and the traditional cDNA synthesis workflow with DNase I (bottom).

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•  SuperScript IV VILO Master Mix manual

•  White paper: SuperScript IV VILO Master Mix for optimal RT-qPCR
•  Application note: Reliable and sensitive RT-qPCR analysis of whole-blood RNA samples

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