Real-time cycler: Applied Biosystems 7900HT

Furthermore, the longer templates derived from recDNA and genomic DNAmimic the average native mRNA lengthof about 2 kb better than shorter templates derived from RT-PCR productoroligonucleotides.

High-Capacity cDNA Reverse Transcription Kit - …

Biology Instruments Division: Applied Biosystems recommends Bionexus's  in its

The High Capacity cDNA Reverse Transcription Kit contains all ..

For a full run of 48 samples, the current protocol takes about five hours, with most of the time being incubation steps (e.g., cDNA synthesis takes two hours). Performing 24 similar reactions manually can be done in approximately the same time, but manual handling introduces variation that can be avoided using the automated approach. The automated protocol outperform the manual procedure when it comes to within-experiment correlation between replicates.

cDNA Protocols | Thermo Fisher Scientific

Several methods for the purification of nucleic acids are available today, including ethanol precipitation and spin-column-based methods. These methods generally require manual input, thereby limiting the throughput of the experiment and increasing variability. In order to reduce human input and increase reproducibility, an automated protocol for cDNA synthesis, labeling, and purification has been developed using a dedicated instrument capable of handling superparamagnetic beads. The procedure is outlined in , where the two purification steps are highlighted.

For optimal performance of the RNA-to-cDNA Kit, Applied Biosystems ..

Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

High-Capacity cDNA Reverse Transcription Kit - ZAGENO

Cloned recDNA and genomic DNAare very stable and generate highly reproducible standard curves evenafter a long storage time, in comparison to freshly synthesized RNA.

Product Application First-strand cDNA synthesis from total RNA

The field of gene expression analsysis has evolved dramatically in recent years. With a basis in microarray technology and the ongoing transition into next-generation sequencing technologies, gene expression analysis is a widely used assay. The microarray technology provides a way of obtaining huge quantities of genome and transcriptome data; it has developed from relatively small-scale experiments using in-house platforms and protocols to more robust studies using what are essentially genome-wide commercially manufactured arrays. Today, several commercial and academic platforms exist, with different array manufacturing and sample preparation approaches. A common criticism of the field of global gene expression analysis has been its lack of standardised experimental protocols and well-defined reference studies comparing different platforms and procedures. To address these issues, the Microarray Quality Control Consortium (MAQC) performed a study where relative gene expression measurements, using one and two-colour platforms, were compared within and between platforms, as well as with TaqMan real-time PCR data [–]. The reference data set, based on standardised commercially available RNA sources, was made publicly available, enabling researchers to benchmark new procedures and platforms.

Comparing RT method and priming method - Applied Biosystems

However, identical RT efficiency, as well asreal-time PCR amplification efficiencies for calibration curve andtarget cDNA must be tested and confirmed if the recDNA is to provide avalid standard for mRNA quantification.