cDNA Synthesis for qPCR

Higher cDNA synthesis efficiency of SuperScript IV VILO Master Mix (Figure 2) allows using lower RNA inputs in RT-qPCR workflow, better RT-qPCR sensitivity to work with low-copy targets and degraded RNA, and archiving 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.

Typically, 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.

RNA purification methods are not foolproof and often fail to completely remove gDNA. Amplification of contaminating gDNA can cause a shift in C_t 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).

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).