Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays.We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation

Another look at nagyagite from the type locality, Sacarimb, Romania: Replacement, chemical variation and petrogenetic implications

Extensive compositional heterogeneity is displayed by Pb-Sb-Au tellurides from the type locality at S $\check{\rm{a}}$ c $\check{\rm{a}}$ rîmb. These phases are collectively considered as varieties of nagyágite in the absence of crystal chemical data confirming the presence of distinct, but topologically closely related compounds. Chemical heterogeneity is seen relative to ‘normal’ nagyágite, with close to the ideal composition Pb3[Pb1.8(Sb1.1,As0.1)1.2]Σ3S6 (AuTe2), which is the primary and common type in the deposit. A modified formula, (Pb3S3)[(Pb2−x )(Sb,As,Te b )1+x (S3−y Te y )]Σ6(Au1−z−w Te2+z S w ), accounts for the chemical variation observed. Values of x (0.2 to 1.15) express substitution of Pb by Sb+As for Me2 in sulfosalt modules in the case of Au-depleted and low-Au nagyágite, and by Sb+As+Te b in high-As and low-Pb varieties (b = x+1−(Sb+As) = 0.24 to 0.29). Excess Te compensates for Au deficiency in the telluride layer, with substitution by S also observed; empirical values of z and w are 0 to 0.45 and 0 to 0.32, respectively. Minor substitution of Te for S (y < 0.17) is noted in all varieties except low-Au. These varieties are formed during replacement of the ‘normal’ type as seen in overprinting relationships in those veins reactivated during rotation of the duplex fault-system responsible for vein formation. Replacement is by coupled dissolution-reprecipitation reactions, as indicated by pseudomorphism of one nagyágite type by another in all cases. Variable rates of both molar-excess and -deficit reaction are invoked to explain the observed chemical and textural modifications. Low-Pb nagyágite is also present in zoned platelets where it grows over resorbed cores of ideal composition. Such platelets are instead interpreted as products of self-patterning in a residual precipitate. A marked depletion in the Au content of some nagyágite lamellae is considered to be a diffusion driven Te for Au substitution in the presence of Te-bearing fluid. Replacement of ‘normal’ nagyágite by other varieties can be linked to high fluid acidity, whereas replacement by galena-altaite symplectites relates to changes in the fTe2/fS2 within a narrow domain defined by coexistence of these two minerals. Nagyágite is a mineral with modular crystal chemistry and is able to adjust to variable rates of fluid infiltration by subtle chemical substitutions. The behavior of nagyágite will map and assist coupling between dissolution and precipitation during such reactions.C. L. Ciobanu, N. J. Cook, A. Pring, G. Damian and N. Căprar