RNA Isolation from Cells Overview

  • Extraction and purification of high-quality total RNA from cultured cells in a 96-well format
  • Efficient removal of genomic DNA and other contaminants
  • No centrifugation or vacuum filtration required
  • Ideal for gene expression applications
  • Supports manual processing and full automation methods on Biomek NXP and FXP Lab Automation Workstations
  • Throughput: Two 96-well plates in approximately two hours and 15 minutes on the Biomek FXP

Application: RNA extraction from cultured cells
Downstream Application Techniques: qRT-PCR, Microarray Analysis

Process Overview

1. Start with cultured cells or primary cells 2. Lyse cultured cells with Lysis Buffer and Proteinase K, transfer into new plate 3. Bind Total RNA to paramagnetic beads 4. Separate beads from contaminants, wash with Wash Buffer and Ethanol 5. Add DNase to digest genomic DNA 6. Re-bind RNA to beads with Wash Buffer and remove contaminants 7. Wash the magnetic beads with 70% Ethanol to remove residual contaminants 8. Elute DNA from magnetic particles.

Higher Recovery of Nucleic Acid

The Agencourt RNAdvance Cell v2 extraction method routinely produced higher total RNA recovery in comparison to competitor methods RNeasy* and MagneSil* from the same number of cells (Figure 1).

Figure 1. Total RNA from 5x104 HeLa cells was isolated using the Agencourt RNAdvance Cell v2 system, RNeasy kit, or the MagneSil kit. Quantitation was performed by RiboGreen* assay. Agencourt RNAdvance Cell v2 consistently produced greater yield than competitors RNeasy and MagneSil.

Quality RNA Purification

The Agencourt RNAdvance Cell v2 system was compared against RNeasy and MagneSil kits for total RNA quality. Analysis of the samples using the Agilent* 2100 bioanalyzer showed that Agencourt RNAdvance Cell v2 isolated RNA had consistently higher RNA Integrity Number (RIN) scores than competitive technologies (Figure 2). For more information on RIN RNA quality scores refer to Genomics and Proteomics v.4; no 5, pp.14– 21.

Figure 2.  RNA samples were isolated using the Agilent Bioanalyzer 2100 RNA NanoChip. Total RNA was extracted from 5 x 104 HeLa cells using (A) Agencourt RNAdvance Cell v2, (B) RNeasy, or (C) MagneSil (standard protocols). The average RNA Integrity Number (RIN) as determined from Agilent 2100 bioanalyzer (five replicates of extraction performed on 5 x 104 HeLa cells for each kit).

Summary

Agencourt RNAdvance Cell v2 is a simple and highly efficient method for isolating and purifying total RNA from cultured cells. Its superior performance delivers high quality RNA for use in microarray or real-time PCR gene expression applications. With flexibility from manual to fully automated 96-well plate formats, the Agencourt SPRI paramagnetic bead-based technology enables efficient removal of contaminants without the need for filtration or centrifugation. Agencourt RNAdvance Cell v2 produces higher recovery, better quality, and more consistent results compared to other available RNA isolation and purification technologies with a walk-away automation solution.