RNA ISOLATION

This protocol automates RNA isolation from bacterial cultures and mouse liver tissue samples using magnetic bead-based technology on the Transcriptic platform.

OVERVIEW

Isolation of high quality RNA is critical for successful downstream molecular techniques such as RT-PCR, construction of cDNA libraries and transcriptome analysis using next-generation sequencing. This high-throughput protocol is designed to provide consistent, efficient and high-quality RNA recovery from bacterial cultures as well as mammalian tissue samples using magnetic bead-based technology on the Transcriptic platform.

METHODOLOGY

The RNA isolation workflow is a flexible, reproducible and high-throughput (up to 96 samples at a time) assay executed on the KingFisher device to process input samples ranging from bacterial suspension cultures to mammalian liver tissues. This protocol allows most steps to be executed at room-temperature per manufacturer’s specifications. During cell lysis, bacterial cultures are treated with lysozyme and frozen mammalian tissues are processed into smaller pieces. Next, samples are lysed in a solution containing RNAse inhibitors followed by binding to magnetic particles. The magnetic particles (0.5-1um) contain a paramagnetic core that allows the particles to move in response to a magnetic field and an outer core that allows it to bind RNA. After several washes and recapture of the magnetic particles, the RNA bound to these particles is eluted and the particles removed. After RNA isolation, the RNA quality is assessed either by the 260/280 absorbance ratios on the NanoDrop Lite spectrophotometer (Thermo) or the RNA integrity number (RIN) using a Bioanalyzer. This protocol captures the entire RNA isolation workflow from sample prep to pure extracted RNA.

RESULTS

To generate bacterial cultures, 150ul of 16-hour cultures of E. coli grown in a 96-well plate were lysed with lysozyme and RNA was isolated using a KingFisher-based magnetic bead-based protocol (MagMAX™ Total Nucleic Acid Isolation Kit).  RNA quality (260 nm / 280 nm absorbance ratios or the RNA integrity) was determined using NanoDrop Lite and the Bioanalyzer instruments. To generate liver tissue extracts, tissue biopsies of whole mouse livers were homogenized and RNA isolated using a KingFisher magnetic bead based protocol (MagMAX™ Total Nucleic Acid Isolation Kit).

Figure 1 Optical density readings at 600 nanometer and RNA yield from 6 samples (150ul each) in a 96-well plate following 16 hours of growth and orbital shaking at 37°C. (A) Absorbance readings on the Tecan Infinite M200 Pro showing bacterial growth in wells A1, B1 and C1 with negative controls (media only) in wells D1, E1 and F1. (B) RNA concentration (ng/ul) show consistent recovery from each well ranging from 10-20 ng/ul with no RNA recovered from wells containing media-only. The RNA quality was high and ranged between 1.9-2.1 based on 260/280 absorbance ratios on the NanoDrop Lite. 

Figure 1 Optical density readings at 600 nanometer and RNA yield from 6 samples (150ul each) in a 96-well plate following 16 hours of growth and orbital shaking at 37°C. (A) Absorbance readings on the Tecan Infinite M200 Pro showing bacterial growth in wells A1, B1 and C1 with negative controls (media only) in wells D1, E1 and F1. (B) RNA concentration (ng/ul) show consistent recovery from each well ranging from 10-20 ng/ul with no RNA recovered from wells containing media-only. The RNA quality was high and ranged between 1.9-2.1 based on 260/280 absorbance ratios on the NanoDrop Lite. 

Figure 2  RNA yield and quality from 20 representative mouse liver biopsies processed in a 96-well plate. The RNA quality was high and ranged between 1.9-2.1 based on 260/280 absorbance ratios on the NanoDrop Lite.

Figure 2  RNA yield and quality from 20 representative mouse liver biopsies processed in a 96-well plate. The RNA quality was high and ranged between 1.9-2.1 based on 260/280 absorbance ratios on the NanoDrop Lite.