50X concentrated qScript One-Step Reverse Transcriptase - Optimized 50X formulation of recombinant MMLV reverse transcriptase for one-step RT-PCR. One-Step SYBR Green Master Mix (2X) - 2X reaction buffer containing dNTPs, magnesium chloride, AccuStart Taq DNA polymerase, stabilizers, and SYBR Green I dye Nuclease-free water
qScript One-Step Reverse Transcriptase (50x)
One-Step Master Mix 2x (optionally containing ROX or Low ROX)
iScript cDNA Synthesis Kits : iScript Reverse ..
As real-time PCR assays have become routine in diagnostic laboratories and because of their prominence, there has been rapid growth of commercially available reagent systems and detection platforms which greatly increase the options available to laboratories. In this study, we evaluated several commercial master mixes in order to identify those which produce the best results using one-step and two-step real-time RT-PCR for the determination of EBLV-1. Five different systems for the generation of cDNA, five two-step PCR kits, and five one-step qRT-PCR kits were compared. The analysed performance evaluation criteria included the generation of a standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. The goal of this study was also to determine if the one-step and two-step systems had the same reaction efficiencies for the detection of EBLV-1 and if one system was more sensitive than another using two real-time thermocyclers.
QScript MicroRNA CDNA Synthesis Kit 100 Rxn
An optimized reagent system that provides highly-sensitive reverse transcription of small single-stranded RNA into 5'-labeled universal cDNA using either total RNA or miRNA-enriched template enabling seamless qPCR analysis
How can I increase cDNA yield from reverse transcriptase PCR?
(i) Rotor Gene Q MDx. Of the five PCR kits tested in combination with the reverse transcriptases provided by the same manufacturer, one RT-qPCR assay (SuperScript Vilo in combination with qPCR Express kit) yielded greater sensitivity and showed a detection of at least 10copies/µL (). All amplicons investigated with the two-step method using qPCR kits and RT kits from the same provider showed satisfactory efficiency of amplification (100 ± 10%). The y-intercept for all tested kits varied between 34.73 and 39.55.
How can I increase cDNA yield from reverse transcriptase PCR
The objective of this study was to evaluate the sensitivity, efficiency, and reproducibility of one-step and two-step systems for the detection of EBLV-1. This virus species was selected for this study for the generation of in vitro RNA transcript. This bat virus is widely isolated on European bats, the vast majority of rabies cases in bats in Europe being attributed to EBLV-1. The increased interest in bats as a potential host for zoonotic viruses has resulted in the detection of many new Lyssavirus species in the past ten years . In our study, we selected five different cDNA generation systems and ten commercial kits, that is, five one-step kits and five two-step kits. These kits are commonly used by National Reference Laboratories that conduct rabies diagnosis (data not shown). Our results demonstrated that there was little difference in reaction efficiency, R2 values, or intra- and interreproducibility between one-step and two-step methods. In our experience, regardless of the real-time cycler used, one-step qRT-PCR showed better limit of detection at 95% than the optimised two-step assays using pan Lyssavirus primers.
Another route may be to change up the cDNA amplification kit ..
In addition to assay performance criteria, another goal of this study was to compare the performance of each kit with the cost, the practicability of each tested master mix, and the presence or absence of Primer-Dimer for low DNA concentrations. Interestingly, the one-step QuantiTect SYBR Green RT-PCR kit, which was shown the more efficient one-step kits, is also one of the most expensive and required the longest thermocycling time while the combination of SuperScript VILO reverse transcriptase and the qPCR Express kit which was found to be the most efficient of the two-step kits using the two thermocyclers is also the most expensive of the two-step kits. No difference in run duration was shown between the best two-step kits, SuperScript VILO/qPCR Express and Maxima H Minus RT/qPCR Maxima, and the one-step kit, QuantiTect SYBR Green RT-PCR, confirming that all the kits are suitable for RNA detection, regardless of the thermocycling times. However, when using the thermocycler Rotor Gene MQDx, the real-time one-step SYBR Green assays with universal pan Lyssavirus revealed a high presence of Primer-Dimers for three out of five one-step kits tested especially when RNA concentration was low (below 100copies/µL) compared to two-step kits (data not shown). The results of such techniques should be interpreted with attention, since interpretation can be difficult, especially for low concentrations of samples.