WHITE PAPER SuperScript IV VILO Master Mix SuperScript IV VILO Master Mix for optimal RT-qPCR Abstract The Invitrogen™ SuperScript™ product line provides market-leading reverse transcriptases (RTs) for the highest quality and reliability in cDNA synthesis. The newest enzyme in this family, Invitrogen™ SuperScript™ IV RT, is a thermostable enzyme that maintains the properties of high processivity and sensitivity even with challenging RNA samples. The new Invitrogen™ SuperScript™ IV VILO™ Master Mix is a first-strand cDNA synthesis master mix designed to transcribe from variable input of RNA to produce linear output of cDNA in two-step reverse transcription quantitative PCR (RT-qPCR). In this study, SuperScript IV VILO Master Mix was compared to the current Invitrogen™ SuperScript™ VILO™ Master Mix and other commercial cDNA synthesis products in an extensive variety of RT-qPCR applications. The results show that SuperScript IV VILO Master Mix delivers the highest yields, fastest reaction speed, increased sensitivity, and better performance with challenging RNA samples while maintaining high linearity and low variability across a broad range of input RNA amounts, giving researchers high confidence in their RT-qPCR results. Introduction Although the enzymatic properties of standard RTs, such as the RT from Moloney murine leukemia virus (M-MLV), have been adequate for basic reverse transcription applications, these enzymes do not offer high yields, sensitivity, speed, thermostability, or tolerance of challenging RNA samples. In RT-qPCR applications, the RT enzyme is not the only consideration for reliable results; the format of the kit used for cDNA synthesis is also important. For example, a one-tube master mix format reduces pipetting steps and handling of samples, reducing contamination concerns and improving reproducibility. An optimized master mix with a robust RT enzyme would improve RT-qPCR data consistency and subsequently reduce the number of repeat trials needed. The original SuperScript VILO Master Mix was introduced to alleviate RT-qPCR analysis inconsistencies from variable RNA input; however, this master mix does not perform optimally with more challenging RNA samples and does not offer fast reaction times. SuperScript IV RT (view the white paper) is a newly designed enzyme that allows very short reaction times, has improved sensitivity, and can perform well even with more challenging RNA samples (degraded or inhibitor-containing samples). Here we describe the benefits of combining SuperScript IV RT and the convenient 5X master mix VILO format for firststrand cDNA synthesis in two-step RT-qPCR applications. Where concern over genomic DNA (gDNA) contamination may be an issue, Invitrogen™ SuperScript™ IV VILO™ Master Mix with ezDNase™ enzyme allows optional gDNA removal. ezDNase enzyme is a novel DNase specific for doublestranded DNA that provides the fastest and gentlest method to remove gDNA from an RNA sample before cDNA synthesis. The results presented here demonstrate that SuperScript IV VILO Master Mix, used with both Applied Biosystems™ TaqMan® and SYBR™ Green qPCR assays, exhibits the best linearity, speed, sensitivity, and tolerance of challenging RNA samples, compared to SuperScript VILO Master Mix and other commercial cDNA synthesis master mixes. With its superior properties, SuperScript IV VILO Master Mix is the optimal choice for cDNA synthesis, helping to ensure the most accurate

Table 1. Reverse transcription reaction components and conditions by supplier. Number of separate master mix components to be mixed (except RNA and water) Reaction setup Reaction cycle times Total cDNA synthesis reaction time SuperScript IV VILO Master Mix Random primers, oligo(dT) primers SuperScript IV VILO Master Mix with ezDNase enzyme 3 (ezDNase enzyme, ezDNase buffer, 5X master mix) Random primers, oligo(dT) primers ezDNase enzyme, ezDNase buffer, RNA, water (2 min, 37°C); add 5X master mix SuperScript VILO Master Mix Random primers Random primers, oligo(dT) primers Random primers, oligo(dT)...

Materials and methods • SuperScript IV VILO Master Mix (Cat. No. 11756050) ––Components: SuperScript IV VILO Master Mix (5X), SuperScript IV VILO Master Mix no-RT control, nuclease-free water • SuperScript IV VILO Master Mix with ezDNase enzyme (Cat. No. 11766050) –– Components: SuperScript IV VILO Master Mix (5X), SuperScript IV VILO Master Mix no-RT control, ezDNase enzyme, ezDNase buffer (10X), nuclease-free water RNA purification RNA was extracted from frozen human lung tissue and purified using the Invitrogen™ PureLink™ RNA Mini Kit (Cat. No. 12183018A) according to the standard protocol....

More cDNA faster than with other commercial kits, even at higher reaction temperatures The high processivity of SuperScript IV RT in SuperScript IV VILO Master Mix allows completion of the RT reaction step in only 10 min. While some other commercial RT mixes also have relatively short protocols (less than 25 min), the short total reaction time does not guarantee better yield. When SuperScript IV VILO Master Mix was compared to SuperScript VILO Master Mix and 9 other commercial cDNA synthesis kits, SuperScript IV VILO Master Mix produced the highest cDNA yields for 14 targets in TaqMan assays...

SuperScript IV VILO Master Mix SuperScript VILO Master Mix Reaction temperature Reaction temperature Reaction temperature Figure 3. Higher thermostability of SuperScript IV VILO Master Mix compared to other commercial kits. of RT enzymes, SuperScript IV VILO Master Mix, SuperScript VILO Master Mix, and two other suppliers’ master mixes were compared using several reaction times (10 min, 20 min, and 60 min) and temperatures (42°C, 50°C, and 55°C) (Figure 3). When the reaction temperature was raised to 55°C, the Ct values increased significantly for the other master mixes, indicating these RT enzymes...