RT-PCR is widely used for measuring gene expression in tissue samples or cell culture systems. Traditionally, it is performed in two separate reaction steps. First-strand cDNA is reverse-transcribed from total RNA or poly (A)+ RNA using a reverse transcriptase. Next, the cDNA is amplified by PCR using a DNA polymerase in another reaction.
The MCLAB's HoTaq™ SYBR Green One-Step RT-PCR Kit allows researchers to perform SYBR Green based RT-qPCR in a single step within a single tube. The kit utilizes our own proprietary engineered QuantumScript HD reverse transcriptase and hot-start Taq DNA polymerase. No additional reagents or steps are required once the reaction is initiated. The kit enables the quantification of specific RNA targets with high sensitivity, unparalleled convenience, and wide dynamic range.
The technique reduces the risk of cross-contamination and minimizes the use of reagents. This method is particularly useful for applications in which the expression of a small number of genes that must be analyzed in many different total RNA samples, and robustly amplifying high-abundance transcripts from crude total RNA preparations.
Gene Expression Analysis, Genotyping, Real-Time PCR
To achieve the best performance, appropriate software, such as ABI Primer Express™, should be used.
Recommended Reaction Conditions:
One cycle at 50°C for 10 to 30 minutes *;
One cycle at 95°C for 10 minutes;
Followed by 40 cycles of: 95°C for 15 seconds, 60°C for 1 minute #;
4°C hold (optional)
* Reaction time could be adjusted according to RNA input.
# Cycle number and annealing temperature are experiment dependent.
Recommended Storage Condition: -20°C