Direct detection of green fluorescent protein messenger RNA expressed in Escherichia coli by rolling circle amplification

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

“…49 The apparent detection level of RNA by RPRCA is approximately 10 9 copies/mL. 21 This detection level is much lower than the actual expression levels of verotoxin 1 and 2 (vtx1 and vtx2) of E. coli O-157 under a certain culture condition in our lab, which involves 4.9 × 10 7 and 1.6 × 10 7 copies/mL (data not shown), respectively. This suggests that the detection level of RPRCA should be improved to 10 6 copies/mL or less mRNA.…”

“…20 Therefore, a series of treatments including precircularization and successive degradation of the linear ssDNA probe have dramatically improved the reliability of the RCA reaction due to clear negative control. [19][20][21][22][23] A lack of nonspecific amplification in test samples considerably contributes to the accuracy of RCA.…”

“…This type of RCA is called RNA-primed RCA (RPRCA) and, indeed, RPRCA is used for microRNA and small RNA detection without reverse transcription. 21,40,[42][43][44][45][46][47][48] When several circular ssDNA probes are tested in detecting in vitro transcribed GFP messenger RNA (mRNA) as a standard RPRCA procedure, the RPRCA reaction is observed under a certain ssDNA probe (Fig. 4).…”

“…PRRCA could be applied to the detection of mRNA expressed in bacterial cells. 21 Escherichia coli expressing GFP mRNA is used to determine the influence of rRNA and tRNA on RPRCA. Successful RPRCA is observed only when the total RNA from induced cells is used in combination with the circular probe possessing a sequence corresponding to the 3′-end.…”

Chaperone Probes and Bead-Based Enhancement To Improve the Direct Detection of mRNA Using Silicon Photonic Sensor Arrays

“…21 More recently other optical 22,23 and electrochemical 24–27 techniques have been successfully applied to the analysis of in vitro transcribed mRNA and mRNA isolated from cell lines. While these approaches to mRNA analysis provide robust analytical capabilities and high sensitivity, further improvements, particularly in multiplexing capability, time-to-result, and potential amenability to the simultaneous analysis of multiple classes of biomarkers, would be of potential value to the clinical diagnostic community.…”