Protocol combines benefits of TRI Reagent sample lysis with MagMAX magnetic bead-based RNA isolation technology
Procedure is compatible with fresh, flash-frozen, or RNAlater-treated tissues
Streamlined procedure allows high throughput processing using multichannel pipettes or automated platforms
Simple, Fast, and Efficient Protocol
The new MagMAX-96 for Microarrays Kit streamlines RNA isolation without sacrificing RNA quality or quantity. The Spin protocol uses TRI Reagent (see RNA, DNA, and Protein from a Single Sample) for sample lysis (<20 mg tissue or 1 x 106 cells) and MagMAX magnetic beads for RNA purification. Magnetic beads can be placed directly into TRI Reagent lysates for RNA purification, eliminating organic extractions, which enables high throughput processing of lysates by manual, multichannel pipetting or automated liquid handling systems.
The alternative No-Spin protocol for smaller samples (<5 mg tissue or 5 x 105 cells) eliminates a 10 minute centrifugation step, uses a DNase treatment, and permits fully automated sample processing.
Excellent RNA Yield and Integrity
Hi gh quality and yield of total RNA is critical for successful gene expression profiling experiments. RNA quality is assessed by the A260/A280 ratio, the ratio of 28S:18S rRNA peaks, and RNA Integrity Number (RIN). As shown in Figure 1, the MagMAX-96 for Microarrays Spin and No-Spin Procedures both purify RNA with good yield and quality. RIN is based on the entire electrophoretic trace of the RNA sample (1=highly degraded and 10=highly intact). Total RNA purified using MagMAX-96 for Microarrays has high integrity (RIN typically >7.8).
Figure 1. Obtain RNA of High Quality and Yield with MagMAX-96 for Microarrays Total RNA Isolation Kit. RNA was isolated from frozen mouse liver with the MagMAX-96 for Microarrays Kit. Tissue homogenates were processed in quadruplets or octuplets using the Spin procedure (homogenate derived from 10 mg tissue per sample) or the No-Spin procedure (homogenate derived from 5 mg tissue per sample), respectively. Purified RNA (2 l) was quantified using a NanoDrop Spectrophotometer (A, B, and C). The ratio of 28S to 18S rRNA was determined with an RNA LabChip Kit and the Agilent 2100 bioanalyzer.
Successful Microarray Experiments
Total RNA isolated from mouse kidney using MagMAX-96 for Microarrays or the Affymetrix recommended protocol (Protocol A: phenol-based sample lysis followed by glass fiber filter RNA purification) were compared by microarray analysis. Normalized microarray signal intensities obtained from two total RNA samples isolated using MagMAX-96 for Microarrays (Figure 2A) and from total RNA samples purified using MagMAX-96 for Microarrays and Protocol A (Figur e 2B) showed excellent correlation. High Percent Present calls, >60%, were also obtained for total RNA purified using the MagMAX procedures and Protocol A (Figure 2C).
Figure 2. Generate High Quality Microarray Data Using Purified Total RNA from MagMAX-96 for Microarrays Kits. RNA, isolated from frozen mouse kidney (1 g, each sample), was amplified using MessageAmp II-96 aRNA Amplification Kit. The biotin labeled amplified RNA was fragmented and hybridized to an Affymetrix GeneChip Mouse Genome 430A 2.0 Array (total probe set=22,000). (A) Scatter plot of normalized signal intensities of two mouse kidney total RNA samples purified using MagMAX-96 for Microarrays; correlation between samples is 0.999. (B) Scatter plot of normalized signal intensities of two mouse kidney total RNA samples purified using MagMAX-96 for Microarrays and the Affymetrix recommended protocol; correlation between methods is 0.998. (C) Percent Present calls for each sample purified using all three methods; >60% Present calls were obtained for all methods.
MagMAX-96 for Microarrays
The MagMAX-96 for Microarray Kit comes with sufficient reagents for 96 purifications from fresh, flash-frozen, or RNAlater-treated tissue samples in a high throughput format. RNAlater-treated samples must be processed with the Spin protocol. The purified total RNA is suitable for microarray analysis, quantitative RT-PCR, and other downstream RNA applications.
Quoc Hoang, WeiWei Xu, Roy Chris Willis, Angela Burrell, Mangkey Bounpheng, Xiaowei Wang, Xingwang Fang Ambion, Inc.
Cat# Product Name Size 1819 MessageAmp II-96 aRNA Amplification Kit 100 rxns 1839 MagMAX-96 for Microarrays Total RNA Isolation Kit 96 purifications 10050 96 well Magnetic-Ring Stand 1 each