Journal:

Article Title: Both Transcriptional and Posttranscriptional Mechanisms Regulate Human Telomerase Template RNA Levels

doi:

Figure Lengend Snippet: (A) Diagram of the construction of pBabe puro hTERT. An EcoRI fragment from pGRN145, containing the consensus Kozak sequence and the full-length human telomerase catalytic subunit cDNA, was cloned in the EcoRI site of the pBabe puro retroviral construct to form pBabe puro hTERT. (B) Diagram of plasmid construction for the generation of competitor RNA for quantitative RT-PCR (pTRC3+). The original plasmid pTRC3 is pGEM-5zf(+) with hTR cDNA cloned in its SacI site, and the orientation is such that the T7 transcript of this construct is the sense strand. The NarI and StuI sites shown are unique sites in this plasmid. The location of the PCR primers used for RT-PCR assays in this study (F3B and R3C [Table 1]) are shown. A 20-bp fragment was inserted into pTRC3 by ligating the annealed oligonucleotides with pTRC3 linearized with NarI. The oligonucleotides contained an equal number of G · C and A · T base pairs, so that the insertion did not change the G+C content of the PCR product compared to the RT-PCR product of hTR. The resulting plasmid, pTRC3+, was linearized with StuI, and competitor RNA was generated with the T7 MAXIscript in vitro transcription kit.

Article Snippet: The antisense riboprobe was generated with the MAXIscript SP6 in vitro transcription kit (Ambion), using pTRC3 (Fig. B) linearized with Stu I as a template.

Techniques: Sequencing, Clone Assay, Construct, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Generated, In Vitro