Primer extension assays were performed as previously described (Ranish and Hahn 1991 (link)) and with a few modifications described in (Kaplan et al. 2012 (link)). Briefly, 30 µg total RNA purified as previously described (Schmitt et al. 1990 (link)) was used to anneal with 32P-labeld oligonucleotide priming downstream of ADH1 start sites in 15 µl total reaction volume. Reverse-transcription reaction was performed by M-MLV reverse-transcriptase (Fermentas) in the presence of RNase Inhibitor (Fermentas) in 45 µl total reaction volume. Products were precipitated, digested with RNase A, and separated in 8% acrylamide gel made with 19:1 acrylamide:bisacrylamide (Bio-Rad), 1XTBE, and 7M urea, followed by visualization by phosphorimaging (Bio-Rad) and quantification with ImageQuant 5.1 software (GE).
Phosphorimaging
Manufactured by Bio-Rad
The Phosphorimaging system at Bio-Rad is a device designed for the detection and quantification of radioactively labeled biomolecules, such as proteins and nucleic acids, in various experimental samples. It utilizes a phosphor screen to capture the radioactive signal, which is then scanned and converted into a digital image for analysis.
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2 protocols using phosphorimaging
1
Primer Extension Assay for RNA Analysis
2
Primer Extension Analysis of IMD2 TSS
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For primer extension (PE) analysis, RNA was isolated from mid-log phase cells grown in appropriate medium, optionally treated with desired concentration of MPA for indicated time periods. PE analysis was done essentially as described earlier (49 (link)) with modifications described in (4 (link)). Briefly, 30 μg of total RNA was annealed with 32P end-labeled oligo. M-MLV Reverse Transcriptase (Fermentas) was used for reverse transcription, in the presence of RNase Inhibitor (Fermentas). PE products were ethanol precipitated overnight and separated on 8% polyacrylamide gels (19:1 acrylamide:bisacrylamide, Bio-Rad) containing 1× TBE and 7M urea. PE gels were visualized by phosphorimaging (Bio-Rad or GE Healthcare) and quantified using Image Lab software (Bio-Rad). For IMD2 TSS annotation we have considered ‘A’ of the start codon (ATG) as +1, so that bases upstream start at −1, as in our previous publication (4 (link)).
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