OPS Diagnostics, LLC
Ribonucleic acids (RNA) isolated from active cells provide a snapshot of a cell’s status and physiology. The profile of mRNA from cells and tissues can provide important insight into gene expression and its relationship to diseased states. However, for such analyses to be accurate the RNA must be of high quality. Quality RNA is synonymous with RNA that is not degraded. Once liberated from cells, RNA is susceptible to breakdown by nucleases, which makes it incompatible for downstream applications.
One common method of quantifying the quality of RNA is to analyze it on the Agilent 2100 Bioanalyzer. The Bioanalyzer is a lab-on-a-chip method for electrophoretically separating and quantitating nucleic acids. Agilent has developed an algorithm for the Bioanalyzer’s RNA chip that assigns a quality value based on the total RNA profile, including the ratios of ribosomal RNAs (Mueller et al., 2016). Compared to estimating quality based on 28S/18S ratios alone, the Bioanalyzer has been shown to yield more consistent results than horizontal gel analysis (Schroeder et al., 2006).
The analysis of the total RNA profile is used to calculate a RIN, or RNA Integrity Number. The RIN is on a scale from 1 to 10, with 10 representing completely intact RNA and 1 (or NA) representing completely degraded RNA. A RIN above 7 is a good indicator that the RNA can be used for downstream applications, such as NextGen sequencing and microarray analysis. Any gene expression analysis that examines the relative levels of RNA depends on the RNA being of good quality.
RNA that is heavily degraded might provide a misleading picture of gene expression. RNases are the biggest challenge when isolating quality RNA, which is released from cells and tissues upon cell lysis or disruption. RNases can be rendered ineffective by using preservation reagents, such as RNAlater™, and by isolating the RNA under conditions where the RNase is inactive. However, even with such precautions, RNA can still be difficult to isolate intact. In addition to RNase degradation, in our opinion, the sample processing techniques may shear the larger ribosomal subunits and lead to erroneous RIN calculations.
Previous work at OPS Diagnostics demonstrated that bead beating, a very widely used method for sample disruption, can significantly shear DNA during the homogenization process. Genomic DNA, when handled carefully, can be as large as 50 Kb, but it can be significantly fragmented by shearing during bead beating. Homogenization generates fragments that form a normal distribution, primarily between 2 Kb to 7Kb, however smaller and larger fragments can be observed at higher deviations. The larger eukaryotic rRNAs used in the calculation of RINs are 1753 bases (18S) and 3354 bases (28S). The larger 28S rRNA falls with the major distribution of DNA fragments generated by bead beating, thus the possibility exists that the 28S rRNA might be susceptible to shearing.
If the larger 28S rRNA shears during homogenization, it could lead to an artificially low RNA Integrity Number, though the RNA is fragmented, but not degraded. Fragmented total RNA would still represent a complete RNA profile, although discontinuous. Consequently, we investigated the effects of bead beating on RNA quality using OPS Diagnostics’ bead beating processes and a commercial RNA isolation kit. RNA was analyzed using an Agilent 2100 Bioanalyzer to assess RNA integrity.
RNA Source: A mouse Hepatoma cell line was used as a RNA source for all experiments. Each bead beating tube required the equivalent of a small cell culture flask (i.e., 25 cm2 T flask). The cell line was cultured on Delbecco’s Modified Eagle Medium (Sigma D1145) with 10% Fecal Calf Serum (Sigma F2442), 1% glutamine (Sigma G7513), and 1% Penicillin/Streptomycin (Sigma P4458) at 37°C with 5% carbon dioxide. Confluent cells were isolated by detaching cells by treatment with Trypsin, inhibiting the Trypsin with DMEM with 10% FCS, and pelleting cells by centrifugation using a Beckman clinical centrifuge at 700 rpm (82 x g). The cell pellet was resuspended in Qiagen RTL Buffer (Qiagen, 79216) from the Qiagen RNeasy Mini Kit (Qiagen, 74104) and then disrupted as described below.
Cell Disruption: As noted, each disruption tube required 25 cm² of cultured adherent cells. Thus 350 µl Qiagen RTL Buffer was added to the cells harvested from one small cell culture flask (or equivalent). Once the buffer and cells were mixed, lysate was generated by forcing the cells through a 21 gauge needle using a syringe. Like a French Press, the cells expand and rupture when exiting the needle. Though this is an effective method, it is very low throughput. This cell lysate was used three different ways for this analysis. In one approach, lysate was bead beated and then the RNA was isolated. A second approach isolated the RNA and then used the purified sample in bead beating. In the third approach, RNA was purified and used as a non-processed control.
RNA Isolation: Total RNA was isolated using the Qiagen RNeasy Mini Kit. Depending on the experiment, RNA was isolated from the cell lysate prior to or after the bead beating. RNA isolation used a standard spin column protocol where RNA in Qiagen RTL Buffer was bound to a silica spin column, washed with buffers to remove contaminants, and then eluted in 50 µl of nuclease-free water. OPS Diagnostics’ silica spin columns (OPS Diagnostics, SSC 100-01) were substituted for Qiagen spin columns. The eluted RNA was analyzed on a DeNovix DS11 spectrophotometer. Then the samples were analyzed on an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Kit.
Bead Beating: Two strategies were used to assess the effect of bead beating on RNA quality. The first approach involved bead beating the cell lysate (generated using a 21 gage needle) in Molecular Biology Grade Pre-filled Tubes, with either 1.0 mm zirconium beads (OPS Diagnostics, PFMB 1000-100-36) or 100 micron zirconium beads (OPS Diagnostics, PFMB 100-100-12), for 1 minute at 4000 rpm in a HT Mini™ homogenizer. RNA was then isolated from processed samples, measured spectrophotometrically and analyzed on an Agilent 2100 Bioanalyzer using a RNA 6000 Nano Kit.
The alternative approach used purified RNA as the sample for bead beating. Individual RNA samples from cell culture lysate were diluted with 150 µl nuclease-free water and then added to pre-filled tubes. Five different media: 1) 1.0 mm zirconium, 2) 1.4 mm zirconium, 3) 2.8 mm stainless steel, 4) garnet with a 6 mm satellite, 5) mixed media of 100 micro;m zirconium, 1.7 mm zirconium, and one 4 mm silica bead were compared to RNA samples with no bead beating. RNA was disrupted for 1 minute at 4000 rpm in a HT Mini™, centrifuged, and analyzed on the Bioanalyzer as described above.
Previous experimentation has demonstrated that bead beating can significantly reduce the length of genomic DNA fragments. With higher molecular weight DNA, smaller beads are more likely to shear the nucleic acid. Conceptually, this same fragmentation could happen to RNA. As molecule size is one of the factors used to calculate RNA Integrity Numbers, assessing the effect of bead beating on RIN was considered a worthwhile exercise. The potential shearing of the larger 28S rRNA could have an impact on whether total RNA preparations are considered suitable for gene expression analyses.
The cell lysate control (no bead beating) had a RIN of 9.9 and a 260/280 ratio of 2.04 (Fig.1). Bead beating with 100 micron and 1.0 mm zirconium beads generated RINs of 9.8 and 10.0 and 260/280 ratios of 2.03, respectively (Fig. 2 and Fig.3). Results from bead beating were in-line with those generated for the control sample, suggesting the bead beating with both small and large beads had no effect on RNA quality.
Cell lysate control RNA, no bead beating
Concentration: 137 ng/µl
260/280 Average: 2.04
100 µm Zirconium
Concentration Average: 110 ng/µl
260/280 Average: 2.03
1.0 mm Zirconium
Concentration Average: 165 ng/µl
260/280 Average: 2.03
In order to directly assess the impact of different grinding matrices on RNA quality, purified RNA was homogenized with a range of different bead types and then analyzed. In this instance, purified RNA with a known RIN of 10 was diluted and added to pre-filled disruption tubes, processed, and then analyzed on a Bioanalyzer. In all cases, the bead beating did not affect the RIN, with only the mixed matrix media (a combination of 100 micron, 1.7 mm, and a 4 mm glass bead) having a slightly lower RIN (Figs. 4-9). This experiment verifies that bead beating does not significantly alter the quality of the RNA.
|Figure 4: Bioanalyzer run on RNA Nano chip, sample was cell lysate.||Figure 5: Bioanalyzer run on RNA Nano chip, sample was cell lysate bead beated in mixed media.|
|Figure 6: Bioanalyzer run on RNA Nano chip, sample was cell lysate was bead beated in garnet.||Figure 7: Bioanalyzer run on RNA Nano chip, sample was cell lysate was bead beated in 2.8 mm stainless steel.|
|Figure 8: Bioanalyzer run on RNA Nano chip, sample was cell lysate was bead beated in 1.4mm Stainless steel.||Figure 9: Bioanalyzer run on RNA Nano chip, sample was cell lysate was bead beated in 1.0 mm Zirconium.|
Both experimental approaches used to assess the impact of bead beating on RNA quality have shown that there is no measureable impact of bead beating on RNA Integrity Numbers. This determination is based on the RIN calculation made by the Agilent 2100 Bioanalyzer software on cell culture lysates.
Mueller, Odilo et al. “ RNA Integrity Number (RIN) – Standardization of RNA Quality Control,” Agilent Application Note, Publication Number 5989-1165EN (2016). Web. 13 Sept. 2016.
Schroeder, Andreas, Odilo Mueller, Susanne Stocker, Ruediger Salowsky, Michael Leiber, Marcus Gassmann, Samar Lightfoot, Wolfram Menzel, Martin Granzow, and Thomas Ragg. “The RIN: An RNA Integrity Number for Assigning Integrity Values to RNA Measurements.” BMC Molecular Biology 7 (2006): 3.PMC. Web. 14 Sept. 2016.