Whatever your requirements, there is a Bio-Rad cDNA synthesis kit that meets your needs.
cDNA Synthesis Kit
Use the iScript™ cDNA synthesis kit for fast, unbiased first-strand cDNA synthesis from both abundant and low-abundance targets.
Reverse Transcription Supermix for RT-qPCR
Choose iScript reverse transcription supermix for fast, efficient, and sensitive cDNA synthesis for RT-qPCR — single tube and a 40 min protocol.
Advanced cDNA Synthesis Kit for RT-qPCR
The iScript Advanced cDNA synthesis kit has an extended dynamic range with a capacity for up to 7.5 μg input RNA.
cDNA synthesis is the first step for many protocols in molecular biology, notably gene expression analysis using real-time quantitative PCR (qPCR). All subsequent steps require that the synthesis step produce cDNA with high fidelity, accurately representing the target DNA. The advent of cDNA synthesis kits has made reverse transcription easier and faster with reproducible high quality. Two important considerations for choice of a kit for cDNA synthesis are the type of primers and the reverse transcriptase. Less-than-optimal priming can lead to over-representation of cDNA synthesized from the 5' or 3' ends of the RNA (5' or 3' bias) or to an abundance of truncated cDNA transcripts.
In many applications, oligo(dT) or random primers can be used to prime cDNA synthesis. Oligo(dT) primers are used to amplify mRNA. However, use of oligo(dT) results in referential priming at the 3' end of the RNA. If the RNA has strong secondary structure, premature termination can result in significantly fewer full-length copies than when priming occurs nonpreferentially at both ends. Additionally, the presence of longer mRNA template increases the probability that transcription will not proceed all the way to the 5' end, leading to an under-representation of 5' sequence.
Random primers are useful for synthesizing cDNA from input RNA because all classes of RNA are amplified including targets such as rRNA, tRNA, bacterial RNA ,or RNA samples that may have some degradation, such as fixed tissue. A disadvantage of random primers is that they can lead to an overestimation of copy number in standard qPCR due to internal priming, which results in short, truncated sequences, which then become over-represented in subsequent rounds of amplification. Droplet Digital™ PCR, which partitions the sample into a large number of individual reactions prior to second-strand synthesis, does not lead to this overestimation.
Combining oligo(dT) and random primers is effective in overcoming the disadvantages associated with each mechanism of priming. In the right ratio the combination provides much better representation of full-length product. Bio-Rad has optimized the blend of oligo(dT) and random primers in its cDNA synthesis kits to increase the proportion of full-length product and minimize 5' or 3' bias.
cDNA synthesis using sequence-specific primers gives the highest specificity. However, a cDNA synthesis reaction generated by specific primers cannot be used for other products. Sequence-specific primers can be used for one-step cDNA synthesis, whereas oligo(dT) and random primers are used in two-step RT-PCR.
Reverse transcriptases have several activities. The RNA-dependent DNA polymerase activity is responsible for first-strand cDNA synthesis. Reverse transcriptase also has some DNA-dependent DNA polymerase activity. A third activity is nuclease activity including RNase H, which degrades RNA when it is present in an RNA-DNA hybrid molecule.
Every Bio-Rad cDNA synthesis kit contains Moloney murine leukemia virus (MMLV) reverse transcriptase, whereas other cDNA synthesis kits use avian myeloblastosis virus (AMV) reverse transcriptase. MMLV has several advantages over AMV, most importantly a lower error rate. The RNase H activity of MMLV is lower than for AMV. Though RNase H is necessary to degrade the RNA template after first strand cDNA synthesis, it has been suggested that a high RNase H activity can result in product degradation leading to fewer full-length cDNA strands, particularly with long extension times. Bio-Rad cDNA synthesis kits also contain potent RNase inhibitors to prevent RNA degradation by the exogenous RNase activities that are ubiquitous in nature.