Journal:

Article Title: Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency

doi: 10.1261/rna.134706

Figure Lengend Snippet: TLC1 levels are constant throughout the cell cycle, depressed at saturating cell density, and variable depending on tlc1 allele and plasmid type. (A) FACS analysis of wild-type haploid cells following release from a 3-h arrest in 5 μM α-factor. “1C” indicates haploid DNA content. At each time point shown, cells were removed from culture and either fixed for FACS anlaysis of DNA content or flash-frozen for RNA isolation. The RNA samples were reverse-transcribed using random nonamers and oligo(dT). The resulting cDNA was amplified with three PCR primer sets: p38–39 to quantify TLC1 total, p90–91 to quantify TLC1 3′ extension, and p34–35 to quantify U2. The concentration ratio of TLC1/U2 at each time point was then divided by the concentration ratio of the prearrest sample, i.e., (TLC1/U2)x/(TLC1/U2)pre-arrest, where x is each time point. (B) Graphical representation of TLC1total RNA levels (top) and TLC13′ extension RNA levels (bottom) at the time points shown in A. Each point on the graphs is the mean of two RT-PCR reactions, and the error bars show standard deviation. At each density point, RNA was isolated and reverse-transcribed using random nonamers and oligo(dT). The cDNAs were amplified with the following PCR primer sets: p38–39 to quantify TLC1 total, p32–33 to quantify ACT1 exon, and p34–35 to quantify U2. (C) Graphical representation of normalized TLC1total RNA levels (top) and ACT1 mRNA levels (bottom) at the cell density points shown below the graph. (D) Northern blot analysis of TLC1 RNA over a cell density course. RNA was isolated from the same strain as in C but from an independent cell density course. Note that U2 is used for normalization in real-time RT-PCR experiments because the U2 RNA (1.175 kb) is similar in size to TLC1 (1.167 kb), while U1 is used for normalization in Northern blots because its smaller size (0.568 kb) permits adequate separation from TLC1. (E) Relative TLC1 RNA levels in strains with different genetic contexts or different tlc1 alleles. WT N indicates wild-type haploid; (CEN), low-copy CEN plasmid; (2μ), high-copy 2-micron plasmid; (dex), dextrose media; (raf), raffinose media; (gal), galactose media; WT 2N, wild-type diploid; and Het 2N, TLC1/tlc1Δ heterozygous diploid. Total TLC1 was normalized to U2 as described in Materials and Methods, then the TLC1/U2 concentration ratio of each sample was divided by the concentration ratio of the WT N sample, i.e., (TLC1tot/U2)x/(TLC1tot/U2)WT N, where x is each strain. The TLC13′ extension was normalized to TLC1total, and the resulting percentage of total TLC1 that contains the 3′ extension is shown above each bar. Note that the Y axis is split; the bottom bar extends from 0 to 1, while the upper bar extends from 1 to 27.5.

Article Snippet: To determine the concentration ratio of TLC1 to U2 in a total RNA sample, we used a reverse transcription mix of random nonamers and oligo(dT) primers (non/dT, Qiagen) to generate an inclusive cDNA pool.

Techniques: Plasmid Preparation, Isolation, Reverse Transcription, Amplification, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Northern Blot, Quantitative RT-PCR