SuperScript VILO cDNA Synthesis Kit and Master Mix
|The Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit and Master Mix are designed to increase cDNA yields and improve the dynamic range of your qRT-PCR assays. This means that you obtain the same relative representation in your cDNA, irrespective of the abundance level of your gene of interest. SuperScript VILO Master Mix is available in a convenient single-tube format.|
The SuperScript VILO cDNA Synthesis Kit is designed to deliver consistent RT results and unprecedented linearity across the broadest range of input materials.
- For probe-based two-step qRT-PCR, linear cDNA yields from 1 pg to 2.5 µg of total RNA in 20 µL reactions
- For Invitrogen™ SYBR™ GreenER™ Two-Step qRT-PCR, linear cDNA yields from 1 pg to 100 ng of total RNA in 20 µL reactions
Reaction volumes can be scaled up to 100 µL if more cDNA is needed, while maintaining the same linearity of the reaction.
Excellent linearity in your RT step means you will:
- Obtain the same relative representation in your cDNA qPCR template, regardless of gene abundance
- During normalization, get both your gene of interest and the reference gene in a linear phase for cDNA synthesis even if their abundances differ in the starting material
- Get greater accuracy in your qRT-PCR data by reducing this common bias
Reliable performance across an extended linear range of RNA input
Figure 1. The SuperScript VILO cDNA Synthesis Kit provides reliable performance across an extended linear range of RNA input. Serial dilutions of total RNA from HeLa cells were reverse transcribed using the SuperScript VILO cDNA Synthesis Kit, followed by triplicate qPCR reactions using human β-actin Applied Biosystems™ TaqMan® assays with the Invitrogen™ EXPRESS qPCR SuperMix Universal on an Applied Biosystems™ 7900HT real-time PCR system. The standard curve (A) shows that even outside the recommended input of 1 pg to 2.5 μg, the kit exhibits a coefficient of correlation of 0.996 for up to 5 μg of input RNA. The amplification plot (B) illustrates that the triplicates are aligned across all dilutions, demonstrating the robustness of the SuperScript VILO cDNA Synthesis Kit over a broad linear range of RNA input. Normalize your lower-abundance genes to your reference genes without worrying about potential variation of RT efficiency at different RNA input levels.
The SuperScript VILO cDNA Synthesis Kit generates far greater yields of cDNA, coupled with reduced downstream qPCR inhibition. This means you can:
- Archive cDNA for future studies
- Use greater amounts of cDNA in qPCR to increase sensitivity up to 4-fold without fear of inhibiting the reaction
This benefit has been achieved through a careful balancing of Invitrogen™ SuperScript™ III Reverse Transcriptase (RT), RNaseOUT™ RNase inhibitor, and the addition of a proprietary helper protein, providing a clear advantage in your qRT-PCR reagents if you have a low-copy target.
|Figure 2. The SuperScript VILO cDNA Synthesis Kit exhibits less PCR inhibition for greater sensitivity in your real-time PCR application. cDNA yield analysis by 32P-dCTP incorporation. Three different kits were compared for cDNA yield, using the same amount of starting HeLa RNA: the Invitrogen™ SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR, SuperScript VILO cDNA Synthesis Kit and MMLV. The VILO kit shows 3- to 4-fold higher cDNA synthesis yield than the other Invitrogen kit and 8- to 9-fold higher than MMLV.|
|Figure 3. The SuperScript VILO cDNA Synthesis Kit demonstrates greater yields than competitors. Total RNA (2 μg and 1 ng) from HeLa cells was reverse transcribed using either the SuperScript VILO cDNA Synthesis Kit (blue) or Mulv competitor kit (green), followed by triplicate qPCR reactions using β-actin–specific TaqMan Assays with the EXPRESS qPCR SuperMix Universal on an 7900HT real-time PCR system. At both high and low RNA input levels, the SuperScript VILOcDNA Synthesis Kit outperformed the Mulv competitor kit by at least 2 cycles, indicating a 4-fold increase in sensitivity.|
Get the most sensitive qPCR performance with the SuperScript VILO cDNA Synthesis Kit regardless of detection method. Using either probe of Applied Biosystems™ SYBR™–based real-time PCR detection, you can expect up to 8-fold sensitivity increases, compared to other commercially available kits. SuperScript VILO cDNA Synthesis Kits are available separately, or as part of our new Invitrogen™ EXPRESS line of qPCR and qRT-PCR reagents designed for fast cycling, real-time PCR instruments.
Figure 4. The SuperScript VILO cDNA Synthesis Kit can be successfully used with SYBR Green–based or fluorogenic probe/primer–based qPCR detection chemistries. (A) Equal amounts (10 pg) of total RNA from HeLa cells were reverse transcribed the SuperScript VILO cDNA Synthesis Kit (blue), Bio-Rad’s iScript™ cDNA Synthesis Kit (yellow), Qiagen’s QuantiTect® Reverse Transcription Kit (green), and Stratagene’s AffinityScript® QPCR cDNA Synthesis Kit (pink). The resulting cDNA was then amplified using hsp70 gene–specific primers in triplicate using the Invitrogen™ SYBR™ GreenER™ qPCR SuperMix on an 7900HT real-time PCR system. The SuperScript VILO cDNA Synthesis Kit exhibits higher sensitivity than the other kits when used with SYBR Green detection, as shown by the Cts that are obtained at least 2 cycles earlier. B. Equal amounts (1 μg) of total RNA from HeLa cells were reverse transcribed using the same kits named in A. The cDNA obtained was then amplified in triplicate TfR gene–specific TaqMan Assays using the EXPRESS qPCR SuperMix with Premixed ROX on an 7900HT real-time PCR system. The SuperScript VILO cDNA Synthesis Kit exhibits higher sensitivity than the other kits when used with probe-based detection, as shown by the Cts that are obtained at least 2 cycles earlier.
SuperScript VILO Master Mix includes optimized buffer, dNTPs, SuperScript III RT, and other reagents, allowing you to perform reverse transcription from a single, convenient mix. Minimize pipetting and, consequently, your risk of contamination, sample mix-ups, and pipetting errors.
Product Literature & Resources
- Product bulletin: SuperScript VILO Master Mix
- How-to video: Making cDNA with the SuperScript VILO cDNA Synthesis Kit
- Webinar: Robust cDNA synthesis with SuperScript III