Q1: What is the minimum/maximum amount of RNA template I can use?
The amount of total RNA required may vary depending on the expression level of the target transcripts. In general, we recommend using 0.1 pg - 5 µg of input RNA.
Q2: Will the blue tracking dye interfere with qPCR fluorophore channels and affect the signal of probe/fluorescent dyes?
The dye used in ZymoScript RT PreMix has been thoroughly tested in downstream analyses. At the provided concentration, it will not affect fluorescent signal readings of any qPCR fluorescent channel.
Q3: How is the performance of ZymoScript RT with low-quality RNA samples?
Lower cDNA yields can be expected with degraded and/or impure RNA samples. We recommend using Quick-RNA or Direct-zol Kits (see Related Products) for high quality RNA extraction.
Q4: Can I use gene specific primers to increase the cDNA synthesis of a target gene?
Yes, gene specific primers can be used to increase specific target yield. However, we recommend using ZymoScript One Step RT-qPCR (see Related Products) for enhanced specificity.
Q5: Can I assemble the RT reaction at room temperature?
Yes, the enzymes are not activated at ambient temperatures.
Q6: Can I store the ZymoScript RT PreMix at Room Temperature or 4°C?
We do not recommend storing the kit at room temperature for prolonged periods of time. The RT PreMix can be stored at 4°C for up to 1 week.
Q7: How can I ensure that there is no genomic DNA contamination in my RT reaction?
Samples can be treated with DNase I to eliminate DNA contamination. DNase I is included in the Quick-RNA and Direct-zol Kits recommended for RNA extraction (see Related Products). Alternatively, the DNase I set can be purchased separately.
Q8: Do I need to add RNase inhibitors?
No, RNase inhibitors are already included in the enzyme mix to prevent unwanted RNA degradation during reverse transcription.
Q9: Can I use the ZymoScript RT PreMix Kit to generate long cDNA for gene/cDNA cloning?
No. The ZymoScript RT PreMix (2X) already contains a mixture of oligo dT and random hexamer primers which is not ideal for the synthesis of long cDNA molecules. For this specific application we recommend using the ZymoScript One-Step RT-qPCR Kit (R3014) with gene-specific primers.
