14 research outputs found
Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation
Abstract C-Repeat Binding Factors (CBFs) are DNAbinding
transcriptional activators of gene pathways imparting
freezing tolerance. Poaceae contain three CBF subfamilies,
two of which, HvCBF3/CBFIII and HvCBF4/CBFIV,
are unique to this taxon. To gain mechanistic insight into
HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in
spring barley (Hordeum vulgare) cultivar âGolden Promiseâ.
The Hv-CBF2A overexpressing lines exhibited stunted
growth, poor yield, and greater freezing tolerance compared
to non-transformed âGolden Promiseâ. Differences in
freezing tolerance were apparent only upon cold acclimation.
During cold acclimation freezing tolerance of the
Hv-CBF2A overexpressing lines increased more rapidly
than that of âGolden Promiseâ and paralleled the freezing
tolerance of the winter hardy barley âDicktooâ. Transcript
levels of candidate CBF target genes, COR14B and DHN5
were increased in the overexpressor lines at warm temperatures,
and at cold temperatures they accumulated to much
higher levels in the Hv-CBF2A overexpressors than in
âGolden Promiseâ. Hv-CBF2A overexpression also
increased transcript levels of other CBF genes at FROST
RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites
in their upstream regions, the most notable of which was
CBF12. CBF12 transcript levels exhibited a relatively constant
incremental increase above levels in âGolden Promiseâ
both at warm and cold. These data indicate that Hv-CBF2A
activates target genes at warm temperatures and that transcript
accumulation for some of these targets is greatly
enhanced by cold temperatures
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 âŒ1,000 copies, seven of which were sensitive to âŒ100 copies, while only 5 were sensitive to âŒ10 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