Journal: mBio

Article Title: tRNAs Are Stable After All: Pitfalls in Quantification of tRNA from Starved Escherichia coli Cultures Exposed by Validation of RNA Purification Methods

doi: 10.1128/mbio.02805-22

Figure Lengend Snippet: Growth state-dependent efficiency and size bias of common RNA extraction methods. (A) Schematic representation of RNA extraction efficiency measurement by [ 14 C]-radiolabeling. Cells were grown in MOPS minimal medium supplemented with [ 14 C]-uracil for approximately two generations before adding nonradioactive uracil. Subsequently, samples were taken during steady-state growth and during valine-induced isoleucine starvation. Samples were spiked in by addition of a small volume of [ 3 H]-labeled cell culture harvested during steady-state growth and either directly subjected to TCA precipitation as an estimate for total RNA or extracted by a selection of common RNA extraction methods. Extracted RNA was analyzed further or precipitated and compared to total, TCA-precipitable RNA to estimate overall extraction efficiency. (B) Overall efficiency of common RNA extraction methods. The efficiency of hot phenol, cold phenol, and modified TRI Reagent extractions was determined as the fraction of recovered RNA out of total RNA. Circles indicate data points of two independent biological replicates; bars indicate the average of the two measurements. Shown is the RNA recovery in three samples obtained during steady-state growth (st-st 1 to 3) and 5, 10, 20, 40, and 80 min after induction of isoleucine starvation. (C to E) Efficiency of RNA extraction from starved cultures relative to steady-state cultures. The relative efficiency of hot phenol (C), cold phenol (D), and modified TRI Reagent (E) extractions was determined as the fraction of recovered RNA after starvation relative to the mean of three steady-state samples. For comparison, the fraction of total, TCA-precipitable RNA relative to the steady state is indicated in blue (dark and light blue for two independent biological replicates). Circles represent data points of two independent biological replicates; dotted lines represent the average of the two measurements. (F) PAGE analysis of radiolabeled RNA obtained by hot phenol, cold phenol, and modified TRI extraction. [ 14 C]-labeled RNA was separated on denaturing 5% polyacrylamide gels and detected by phosphor imaging. Bands corresponding to major RNA species (rRNAs and tRNAs) are indicated in black. Bands attributed to other abundant RNA species (tmRNA/10S RNA, SsrS/6S RNA, and Ffs/4.5S RNA) are indicated in gray. (G to I) Efficiency of extraction of individual RNA species by hot phenol (G), cold phenol (H) and modified TRI Reagent (I) extraction from starved cultures relative to steady-state cultures. RNA bands detected in panel F corresponding to 23S, 16S, and 5S rRNA and tRNAs were cut out and RNase-treated, and the incorporated radioactivity was measured to obtain the [ 14 C]/[ 3 H] ratio for normalization. Filled symbols represent data points of two independent biological replicates; dotted lines represent the average of the two measurements. The dash-dotted line indicates the steady-state level.

Article Snippet: To serve as a spike-in for normalization, a culture of MAS1081 was grown in MOPS minimal medium supplemented with 20 μCi/mL (0.98 μM) [5- H]-uridine (20.4 Ci/mmol; Perkin Elmer) and harvested by transfer to a 1/5 culture volume stop solution.

Techniques: RNA Extraction, Radioactivity, Labeling, Cell Culture, TCA Precipitation, Selection, Modification, Imaging