Quantitative mRNA expression measurement at home

Abstract

mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA. The sensitivity of LAMP in early reports has been below that of the standard RT-PCR tests. Here, we report the use of a fluorescence-based RT-LAMP protocol to measure CDX2 expression patterns, which match extremely well to the standards of sophisticated RT-PCR techniques (r = 0.99, p < 0.001). The assay works on diverse sample types such as cDNA, mRNA, and direct tissue sample testing in 25 min compared to more than 3 h for RT-PCR. We have developed a new protocol for designing RT-LAMP primers that reduce false positives due to self-amplification and improve quantification. A simple device with a 3D-printed box enables the measurement of mRNA expression at home, outdoors, and point-of-care setting.

Figure 1

Workflow for faster measurement of mRNA expression. (A) Schematic displays of the proposed direct sample testing using RT-LAMP for quick-expression measurement in colon tissue. (B) Proposed device and workflow to demonstrate mRNA expression measurement at home.

Figure 2

Rapid CDX2 expression measurement using RT-LAMP and comparison with RT-PCR. (A) Five parameter logistic (5PL) model for comparative analysis of RT-PCR and RT-LAMP data. 5PL model parameters: baseline (a), slope factor (b), inflection point (c), plateau (d), and asymmetry factor € are displayed for a sample RT-PCR (left) and RT-LAMP (right) data. The X-axis displays time in minutes, and the y-axis displays the raw fluorescence intensity. 5PL model is computed using least squares regression, and the model predicted data is shown as a blue line on top of the original raw data as grey dots. The inflection point is shown using a vertical red line. (B) RT-LAMP experiments using hACTB, ACTB2, CDX2, and hCDX2 LAMP primer sets on three different human tissues (lung, colon and blood) cDNA samples. (C) RT-LAMP experiment is performed on cDNA, mRNA, and Tissue quick extract samples. Welch's two sample two-tailed unpaired t-test is performed to compute the p values.

Figure 3

Correlation between RT-PCR and RT-LAMP. (A) Serial dilution was used to prepare human colon cDNA samples in various concentrations. hACTB and hCDX2 LAMP primer sets were used to perform RT-LAMP experiments from the diluted samples. F3 and B3 primers (hACTB and hCDX2) were used to perform RT-PCR experiments on the same diluted samples. The 5PL model was used to identify the inflection point for RT-LAMP and RT-PCR data and visualized using a scatter plot. This experiment was repeated in two human individuals’ cDNA samples (left and right). Correlation tests between RT-PCR and RT-LAMP data for both primers were calculated and displayed as scatter plots using Python seaborn lmplots with the p-values. The confidence interval around the regression line is indicated with shades. (B) 3D-model (4’’ × 3’’ × 5’’) of the 3D-printed box and device assembly details for use at home and outdoor settings. It uses an Arduino Pro Mini microcontroller and ESP32-CAM camera module to detect fluorescence signals. Results are displayed in a 0.96" 128X64 OLED LCD Display. Data from the device is collected over WIFI using an ESP32 module to a cell phone or a computer.