Q1: What is the minimum/maximum amount of RNA template I should 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 the ZymoScript One-Step RT-qPCR Kit has been thoroughly tested in downstream analysis. At the provided concentration, it will not affect fluorescent signal readings during qPCR.
Q3: How is the performance of the ZymoScript One-Step RT-qPCR kit with low-quality RNA samples?
Lower signal can be expected with degraded and impure RNA samples. We recommend using Quick-RNA or Direct-zol Kits (see Related Products) for high quality RNA extraction.
Q4: Can I use oligo dT or random hexamer primers?
No, only use gene specific primers when using ZymoScript One-Step RT-qPCR Kit.
Q5: Can I assemble the RT reaction at room temperature?
Yes, the reaction can be assembled at room temperature. The enzymes are only activated at higher temperatures, ensuring maximum specificity.
Q6: Can I store the ZymoScript One-Step RT-qPCR kit at Room Temperature or 4°C?
We do not recommend storing the kit or kit components at room temperature for prolonged periods of time. The kit 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 or Direct-zol Kits recommended for RNA extraction. Alternatively, the DNase I set (see Related Products) 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: Should I use SYBR Green or a TaqMan probe-based assay?
For single target detection, SYBR Green based assays offer a more affordable solution. For SYBR Green based assays, add 1 µl of Fluor Dye solution (20X) to each 20 µl RT-qPCR reaction. TaqMan probe-based assays are in general more expensive than SYBR Green based assays. However, they present an advantage when multiple targets are analyzed simultaneously and when a superior level of specificity is required. Do not add Fluor Dye solution to the RT-qPCR reaction when performing TaqMan probe-based assays.
Q10: Does ZymoScript One-Step RT-qPCR contain a passive reference dye?
No, a passive reference dye is not included.
Q11: What is the maximal amplicon length that can be achieved using the ZymoScript One-Step RT-qPCR Kit?
Although this product performs the best with relatively short amplicons (50 bp – 1 Kb), it has been successfully used to amplify regions up to 5 Kb long.
