Hiscript Ⅲ RT SuperMix for PCR (+gDNA wiper) is an improved version of Hiscript Ⅱ Q RT Supermix for GPCR(+gDNA wiper), including Hiscript Ⅲ Reverse Transcriptase, a novel reverse transcriptase with optimized Buffer. This kit enhances the efficiency of DNA synthesis and is ideal for two-step qRT-PCR detection.
The 4 x DNA wiper Mix in the kit fully eliminates residual genomic DNA from the RNA template, thus providing more reliable quantitative results. It eliminates the need to design primers across introns, thus simplifying PCR primer design.
5 x Hiscript Ⅲ qRT SuperMix consists of all components needed for the reverse transcription reaction and can be started quickly by adding template RNA and Rnase-free ddH₂O. The gDNA wiper is terminated so that the integrity of the cDNA is not affected. It is compatible with probe-based and dye-based qPCR, allowing high-performance gene expression analysis.
Product advantages
- Simple and quick operation: The 5 × HiScript® III qRT SuperMix comprises all the components needed for reverse transcription. By adding template RNA, a reverse transcription reaction can be completed in 20 minutes
- Broad template compatibility: Compatibility with different species of RNA templates with poor integrity
- High tolerance of impurities: For reverse transcription, the product has a strong tolerance to common impurities such as isopropyl alcohol, ethanol, humic acid, guanidine isothiocyanate, water balance phenol, etc.
- Improved reverse transcription efficiency: In comparison with commonly available reverse transcription products, the HiScript® III RT SuperMix for qPCR (+gDNA WIper) reverses the smallest quantitative CT value of cDNA and has better reverse transcription efficiency
Applications
This product is suitable for the reverse transcription of plant, animal and microbial RNA. The products are compatible with probe-based and dye-based qPCR.
Self-prepared materials
- RNase-free centrifuge tube (1.5 ml), RNase-free tips, RNase-free PCR tube (0.2 ml)
- PCR instrument, pipette, ice or ice box
RNA
High-quality RNA is needed to obtain high-quality cDNA. The user has to verify the RNA integrity by electrophoresis prior to the experiment.
qPCR reagent selection guide
The 1st strand cDNA product can be directly used as the template for gPCR. For PCR, it is recommended that the volume of the template cDNA product should be no more than 1/10 of the total volume of qPCR reaction. ChamQ Universal SYBR qPCR Master Mix (Vazyme #Q711) or AceQ Universal U’ Probe Master Mix V2 (Vazyme #Q513) can be chosen as the qPCR reagent.
Notes
- The 4× gDNA wiper Mix, 5×No RT Control Mix, and 5×HiScriptI qRT SuperMix have high concentration of glycerol. Users should centrifuge briefly and pipette up and down to mix thoroughly prior to use.
- No more than 1 g of total RNA is to be added to the 20 L reverse transcription reaction system. If target genes have low expression levels, the amount of total RNA can be up to 5 g. Otherwise, the amount of RNA added is too high, which may exceed the linear range of subsequent qPCR.
- The cDNA products can be used for qPCR reactions and not for long-fragment PCR amplification of downstream experiments such as cloning. If necessary, HiScript Il 1st Strand cDNA Synthesis Kit(+gDNA wiper)(Vazyme #R312) can be used.
- Reverse transcription can be performed straight away with 5× HiScript I qRT SuperMix, thus avoiding the genome removal step. However, 4× gDNA wiper Mix should not be used with reagents without genome removal module as the Mix in the kits without the latter does not contain the components to terminate the gDNA wiper reaction. This could affect subsequent gPCR results.