Hifair™ AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR _ 11151ES

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Hifair™ AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR represents an enhanced iteration of the Hifair™ Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA Digester Plus). It is designed to fulfill the demands of conducting both reverse transcription and genome removal reactions within a single tube. Moreover, it streamlines the reaction process, significantly reduces reaction time, and augments the yield of the reverse transcription product. The 4×Hifair® AdvanceFast One-Step RT SuperMix encompasses all essential reagents for the reverse transcription reaction. During the reaction, you simply need to supplement it with the gDNA Remover Mix, template RNA, and water to efficiently synthesize the initial strand of cDNA while concurrently eliminating genomic DNA contamination. This product is well-suited for the reverse transcription of intricate RNA templates and proves highly effective for reverse transcription involving scant template amounts and low-copy genes. The reverse transcription product generated by this particular product is fully compatible with both the probe-based and dye-based qPCR methods. For probe-based qPCR, we recommend using Hieff Unicon® Universal TaqMan multiplex qPCR master mix (Cat#11211), while for dye-based qPCR, Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Cat#11184) is the recommended choice for high-performance gene expression analysis.However, it's important to note that the reverse transcription product produced by this product is not suitable for subsequent applications such as the amplification of long fragments. If you require efficient reverse transcription for these purposes, we recommend utilizing the Hifair® AdvanceFast 1st Strand cDNA Synthesis Kit (Cat#11149, Cat#11150). Features 5 mins of RT Integrated RT and gDNA digestion in one tube Applications RT-qPCR Antisense RNA synthesis Specifications Type of end product cDNA (First-Strand) PCR method RT-PCR Type of reagent Reverse Transcription Type of sample RNA Optimal reaction temperature 37℃ Reverse transcriptase M-MLV Components No. Name 11151ES10 (10T) 11151ES60 (100T) 11151-A 4×Hifair® AdvanceFast One-Step RT SuperMix 50 μL 500 μL 11151-B gDNA Remover Mix 10 μL 100 μL 11151-C RNase-free Water 1 mL 2×1 mL Shipping and Storage The product is shipped with dry ice and can be stored at -15℃ ~ -25℃ for 12 months. Instructions for use Thaw the 11151-A/B/C components on ice, gently agitate and thoroughly mix each reagent component. Then, assemble the following components within an RNase-free tube: Component Vol（μL） RNase-free Water To 20 μL 4×Hifair® AdvanceFast One-Step RT SuperMix 5 μL gDNA Remover Mix 1 μL Total RNA 10 pg -5 μg* or mRNA 10 pg-500 ng* Table 1: Reaction preparation *We suggest not exceeding 2 μg as the total RNA input amount. In case of low expression abundance of the target gene, the total RNA input can be increased up to a maximum of 5 μg. Temperature Time 37℃ 5 min* 85℃ 30 s Table 2: Reaction Protocols*This process encompasses both reverse transcription and gDNA digestion.The reverse transcription product is suitable for immediate use in qPCR reactions or can be stored at -20°C for a short duration. For extended storage, we recommend aliquoting and storing it at -80°C to prevent multiple freeze-thaw cycles.Precautions:1. All procedures should be conducted on ice, with utmost care to prevent RNase contamination.2. Ensure your safety and well-being by wearing a laboratory coat and disposable gloves.3. This product is intended solely for scientific research purposes and should not be used otherwise. Figures: High Reverse Transcription Yield Figure 1. Three genes were detected using human 293T cell RNA as a template. Brand A, Brand B, and Brand C are all one-step gDNA removal & reverse transcription reagents. The test results show that with inputs of 1 μg and 10 ng of Total RNA, the reverse transcription yield of 11151ES is superior to other brands, with lower Ct values. Figure 2. Using Arabidopsis RNA as a template, Brand A, Brand B, and Brand C are all one-step gDNA removal & reverse transcription reagents. The test results show that with inputs of 1 μg and 10 ng of Total RNA, the reverse transcription yield of 11151ES is superior to other brands, with lower Ct values. Outstanding Sensitivity Figure 3. Using human 293T cell RNA as a template, genes with 30% GC, 50% GC, 60% GC, and 70% GC contents were amplified. Reverse transcription and quantitative PCR detection were performed using Total RNA inputs of 100 pg, 1 ng, 10 ng, 100 ng, and 1 μg, respectively. The results demonstrate that 11151ES can accommodate RNA reverse transcription across a range of 100 pg to 1 μg. Excellent Stability of cDNA Products Figure 4. Reverse transcription was performed using human 293T cell RNA as a template, and the obtained cDNA products were subjected to repeated freeze-thaw cycles (10, 20, and 30 cycles, respectively). The test results show that the cDNA products generated by 11151ES maintain a high degree of stability even after 30 freeze-thaw cycles, with negligible differences in Ct values. Figure 5. Reverse transcription was performed using human 293T cell RNA as a template, and the obtained cDNA products were stored at -20°C, 4°C, 25°C, and 37°C for 7 days. The test results show that the cDNA products generated by 11151ES maintain a high degree of stability after storage at various temperatures for 7 days, with negligible differences in Ct values. FAQ Q: Does the 11151ES reagent feature color tracing? A: Component A is blue and functions as a sample loading tracker. It is not recommended for downstream colorimetric reactions like LAMP, as it may interfere with color development. Q: Can I skip the gDNA removal step when using 11151ES? A: Yes. If gDNA has already been removed during RNA extraction, you can omit gDNA Remover Mix (Component B) and bring the reaction volume up with RNase-free water. Yes. Scale up the reaction system proportionally according to the manual, and make sure to adjust the reaction volume settings on your PCR instrument accordingly. Documents: Safety Data Sheet 11151_MSDS_HB250721.pdf Manuals 11151_Manual_Ver.EN20250721.pdf Related blog： The Reasonable Range of Ct Values in qPCR Experiments Show more