Highest cDNA yields in the shortest time

Invitrogen SuperScript IV VILO Master Mix is a cDNA reaction master mix designed for two-step quantitative PCR (RT-qPCR) applications. The master mix format elevates the trusted Invitrogen VILO technology (Variable Input, Linear Output) to the next level by combining optimized buffer conditions with Invitrogen SuperScript IV Reverse Transcriptase (RT), known for its high processivity and thermostability.

This new formulation allows the cDNA reaction to occur at higher temperatures with shorter reaction times, resulting in greater cDNA yields and sensitivity even with samples of suboptimal purity and low template levels.


A new tool to enable more efficient and reproducible RT-qPCR

  • Simplified protocol—cDNA synthesis in 10 minutes and optional removal of gDNA in 2 minutes 
  • Higher yields—consistently lowered Ct values by >2 cycles compared to other commercially available RTs 
  • Greater sensitivity—efficient amplification even with samples of suboptimal purity

Video: Overcoming RT-qPCR challenges

SuperScript IV VILO Master Mix (SSIV VILO) includes SuperScript IV RT in an optimized format to give researchers both the convenience and the performance advantages of the robust enzyme for RT-qPCR applications. Compared to other RT enzymes on the market, SuperScript IV RT as a stand-alone enzyme has significantly improved processivity, which consequently leads to greater sensitivity to low-abundance targets, higher resistance to RT reaction inhibitors, and faster reaction times. In the master mix format, with the proprietary helper protein that further improves the interaction between the RT enzyme and the RNA template in an optimized buffer environment, the SSIV VILO has even higher efficiency than SuperScript VILO or any other cDNA synthesis kit.  

In an RT-qPCR analysis of 14 target transcripts using low initial RNA input, SSIV VILO produced the highest efficiency over all other commercially available cDNA synthesis kits tested (Figure 1). SSIV VILO consistently lowered Ct values by >2 cycles with a 10 minute reaction time.  

Figure 1. RT efficiency of SSIV VILO compared to 10 commercial first-strand cDNA synthesis master mixes by RT-qPCR. Master mixes were used to synthesize cDNA in 20 μL RT reactions, per manufacturer instructions, using 1 ng of total HeLa RNA input. For qPCR, 1 μL of RT reactions was used in 10 μL Invitrogen EXPRESS qPCR SuperMix reactions with Applied Biosystems TaqMan® primer and/or probes for gene targets indicated. qPCR results are shown normalized to fold change relative to SSIV VILO [=1/(2^(Ct SSIV VILO – Ct Competitor))].

Survey and qualitative feedback in recent years revealed that researchers are not satisfied with their current cDNA synthesis results and are performing reactions with increasingly difficult samples, such as poorly purified RNA that contains inhibitors or degraded RNA.  To address the performance of reverse transcription with difficult samples, SSIV VILO was compared with other commercially available cDNA synthesis kits for RT efficiency in the presence of a variety of inhibitors.  Ct values generated by kits from other suppliers were normalized to Ct values of SSIV VILO using the equation: Normalized Y values = [1/2^(Ct SSIV VILO -Ct Supplier x)].  As exhibited in Figure 3A, SSIV VILO has the highest efficiency among all cDNA synthesis kits in the presence or absence of inhibitors.  Similarly, SSIV VILO delivers improved performance when frozen samples (Figure 3B) and FFPE samples (Figure 3C) were tested for cDNA synthesis. 

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Figure 3A. SSIV VILO was compared to 8 other commercially available master mixes. RT was performed using 100 ng total HeLa in 20 μL RT reactions plus or minus the concentration of the inhibitors indicated. Performance in qPCR was evaluated in 10 μL reactions with TaqMan primer/probes for the B2M gene targets using EXPRESS qPCR SuperMix.

Get the most sensitive qPCR performance with SSIV VILO, regardless of the detection chemistry you use for your real-time PCR.  Using Applied Biosystems SYBR Green dye–based real-time PCR detection, you can expect an average of 4-fold increase in sensitivity with SSIV VILO as compared to other commercially available kits (Figure 4A).   Similar sensitivity is achieved when using TaqMan probe–based real-time PCR detection chemistry (Figure 4b).  The results are shown with ΔCts of SSIV VILO and 2 other suppliers (Ct suppliers) all compared with SS VILO.  For 92 of 96 targets (96%), SSIV VILO demonstrated lower Ct values than competitors; and for 81 of 96 targets (84%), SSIV VILO resulted in lower Ct values than SS VILO.  The plot shows that for many targets, SSIV VILO is more than 2 Cts lower than that of other suppliers including SS VILO.   

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Figure 4A. Comparison of SSIV VILO with 3 commercially available master mixes (including SS VILO) in a SYBR RT-qPCR gene panel analysis using 100 ng total HeLa in 20 μL RT reaction mixture. Performance in qPCR was evaluated in 10 μL reactions with SYBR primer/probes for the gene targets indicated using EXPRESS qPCR SuperMix.
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Figure 4B. Comparison of SSIV VILO with 3 commercially available master mixes (including SS VILO) in a TaqMan RT-qPCR gene panel analysis using 100 ng total HeLa in 20 μL RT reaction mixture. Performance in qPCR was evaluated in 10 μL reactions with TaqMan primer/probes for the gene targets indicated using EXPRESS qPCR SuperMix.

Many RNA isolation kits and methods do not generate RNA that is completely DNA-free; therefore, gDNA elimination remains a critical step in RT-qPCR.  The presence of residual DNA may lead to false positives, higher background or lower assay sensitivity.  The Invitrogen SuperScript IV VILO Master Mix with ezDNase Enzyme provides a simplified workflow that includes 2 minutes for an (optional) gDNA elimination step and excludes a DNase thermal inactivation or removal step (Figure 5).   Invitrogen ezDNase enzyme, a novel double-strand-specific, thermolabile DNase, is engineered to remove contaminating genomic DNA without affecting quality or quantity of target RNA and without damage to single-stranded DNA such as primers and probes. 

 

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