The AffinityScript Multiple Temperature RT is engineered for improved performance over a broad range of cDNA synthesis temperatures. This multi-temperature capability allows you to change your RT reaction temperature without having to change your reverse transcriptase. You are assured of high cDNA yields from 37°C to 55°C, whether priming at room temperature with random hexamers, or at stringent temperatures designed to enhance priming specificity, or for transcription through GC-rich sequences.
Ideal Sensitivity in Detecting Low Input RNA
When you are working with limited samples, such as small animal models, small organs, or biopsies, our AffinityScript RT is the ideal choice to convert that precious RNA into stable cDNA. Our RT routinely detects and amplifies targets using 1 ng of total RNA.
Two RT-PCR Systems for Cloning or High Sensitivity
The AffinityScript RT is engineered to produce the highest yields of full length cDNA. Whether you are working with genes as small as 500 to 1000 bases or as large as 5, 10, or even 20 kb, the AffinityScript RT is the ideal choice to convert these RNA transcripts into full length cDNA. To demonstrate full length feature, we PCR amplified a 0.6-kb product located at the 5' end of the human nebulin mRNA, which is 20 kb in length, indicating complete reverse transcription of the nebulin mRNA.
See the full experiment in related literature by selecting "posters"
Reverse transcription is the first step in RT-qPCR and thus any inhibition of reverse transcription activity due to the presence of inhibitors could lead to significant error in quantification or result in generation of false negative data. Potential RT inhibitors include chemicals present during RNA isolation (e.g., guanidine, ethanol, formamide, EDTA, SDS) or endogenous substances co-purified during RNA extraction (e.g., plant polysaccharides xylan and pectin, etc.). Furthermore, researchers increasingly use sources of RNA that are less pure, for example formalin fixed paraffin embedded material, or they skip the nucleic acid purification step entirely by using buffers that lyse mammalian cells and tissues and preserve RNA.
We have discovered that AffinityScript RT (E69K, E302R, W313F, L435G, N454K), previously developed for higher thermostability1, is also significantly more resistant to common RT-qPCR inhibitors2 compared to wild-type. As we demonstrate by radioactive incorporation assay, this mutant has much higher tolerance for guanidine isothiocyanate, ethanol, formamide, pectin, and xylan compared to wild-type MMLV-RT and commercially available RNaseH- MMLV-RT mutants (SuperScript II and SuperScript III). Furthermore, we show how inhibition of reverse transcription affects the qPCR results when amplifying in the presence of inhibitors or from crude RNA preparations.
Compared to other RTs, AffinityScript RT tolerates higher concentrations of inhibitors in RNA samples used for RT-qPCR: Results obtained with 50U RT showed the following sensitivity to inhibition by formamide: (lowest) Aff>SuII≥MMLV≥SuIII (highest), which paralleled relative differences in IC50 values for formamide: (>15% v/v) Aff>MMLV≥SuII ≥SuIII (7% v/v). Not surprisingly, inhibition with formamide was overcome to some extent by using 200U SuII and SuIII (ΔCq between 0 and 9% formamide: 50U SuII, 8.1; 200U SuII, 4.1; 50U SuIII, 9.3; 200U SuIII, 4.6). However, higher RT amounts (200U versus 50U) can be inhibitory to RT-qPCR, as shown here by Cq delays of 2.1 (SuII) and 3.9 (SuIII) in the absence of formamide.
For Research Use Only. Not for use in diagnostic procedures