Scheme of work for estimating RNA copy number by determining the efficiency of cell lysis, RNA extraction, RT-qPCR, and FISH imaging. Briefly, the cell number is determined and the efficiency of cell lysis is estimated based on the amount of DNA recovered (total DNA is assayed spectroscopically and individual genes by RT-qPCR) compared with the expected amount of DNA, assuming that in an exponentially growing population of haploid cells the average cell contains 1.4 genomes worth of nuclear DNA. The S. cerevisiae genome is ∼0.012 pg (Fungal Genome Size Database www.zbi.ee/fungal-genomesize). Total RNA purified from the yeast lysates is measured optically. The efficiency of RNA recovery is estimated by measuring the recovery of a known amount of nonyeast RNA (in our case, Arabidopsis thaliana LSM1) added to the lysate. The reverse transcription and qPCR stages are standardized by comparison with known amounts of in vitro transcribed RNAs with the same sequence, mixed with total RNA purified from cells that do not express the reporter genes. The results from this procedure are compared with numbers obtained by visual inspection of individual transcripts detected by FISH in large numbers of single cells. The details of the various procedures are given in the Supplemental Material.
