The specially formulated reaction buffer contains oligo dT and random hexamers and provides unbiased representation and equivalent cDNA abundance over a wide range of input RNA. The high efficiency conversion provides maximum sensitivity for quantification of low abundance RNAs and single-cell expression profiling. The patent-pending formulation captures both 3'-end and 5'-end cDNA sequence and comes in a convenient pre-mixed format that simplifies reaction assembly and generates improved precision and reproducibility in RT-PCR applications. The table below demonstrates the ease of set-up compared to the leading competitor.
Performance Characteristics
Figure 1 demonstrates the high efficiency of cDNA Synthesis. In this experiment, RNA transcripts and cDNA standards were serially diluted and the RNA was then converted to cDNA with the Marligen First-strand cDNA Synthesis System for Quantitative RT-PCR. Comparison of the amplified RNA and the cDNA standard curve demonstrates the high transcription efficiency achieved with the Marligen First-strand cDNA Synthesis System.
Figure 2 shows that cDNA prepared with the Marligen's First-strand cDNA Synthesis System provides significantly enhanced sensitivity compared to cDNA prepared with a competitor's kit. In this experiment, cDNAs prepared using each method were amplified in real time with primers specific for the 5' end of the ADAR gene using three different levels of input RNA. At all three levels of input RNA, cDNA prepared using Marligen’s kit has increased sensitivity in Quantitative RT-PCR.
Quality Testing
For quality testing of these products, first-strand cDNA synthesis is performed on 10-fold serial dilutions from 1 picogram to 1 milligram of total RNA from HeLa S3 cells. One-tenth of each first strand reaction is used as template for SYBR Green real-time PCR of beta-actin mRNA in duplicate reactions. Linearity, sensitivity, and dynamic range are verified by Ct standard curve analysis. All solutions are verified to be nuclease-free.
