Article Title: Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing
Figure Lengend Snippet: Three follow-up experiments attempting to detect three putative RNA editing sites in the C. vulgaris psbI mRNA. ( A ) Chara was grown in nine different environmental conditions: 20 °C, 25 °C, 30 °C and pH 6, 7, 8, in triplicate, and then DNA + RNA were extracted from each. The melt temperatures for genomic DNA (gDNA) and complementary DNA (cDNA) were determined as paired reactions using high resolution melt analysis. Melt temperature differences between gDNA and cDNA derived amplicons were calculated by subtraction. Data presented on these graphs are the averages of nine observations ± standard error (SE). There were qualitative differences in the cDNA–DNA melt temperatures of psbI and the negative control, psbA , at the different incubation temperatures but these differences were statistically insignificant. Different pH also had no significant effect on the cDNA–DNA melt temperatures and two-way analysis of variance detected no interaction between the temperature and pH for either gene; ( B ) RNase H-dependent PCR (rhPCR) was used in an attempt to detect single base changes between the psbI gDNA and cDNA extracted from Chara incubated in different temperatures. This assay was designed to suppress PCR amplification of the genomic sequence but not cDNA made from an edited transcript. The quantitative PCR (qPCR) amplification curves show that gDNA and cDNA amplified with sigmoidal curves with standard PCR but that both gDNA and cDNA were suppressed by the addition of an rhPCR primer despite the presence of RNase H2 in the reactions. This suggests no editing occurred at any of the tested temperatures; ( C ) cDNAs were PCR amplified and sequenced from total RNA extracted from Chara grown at 20 °C, 25 °C, and 30 °C. The sequences all matched the genomic sequence, suggesting no editing had taken place.
Article Snippet: Complementary DNA (cDNA) was produced from DNase treated RNA using Bio-Rad’s iScript cDNA synthesis kit (Hercules, CA, USA).
Techniques: Derivative Assay, Negative Control, Incubation, RNase H-dependent PCR, Polymerase Chain Reaction, Amplification, Sequencing, Real-time Polymerase Chain Reaction