RNAdvance Cell v2 Performance and Data
Total RNA Isolation and Purification from Cultured Eukaryotic Cells
The RNAdvance Cell v2 kit is a ribonucleic acid (RNA) isolation reagent kit built on SPRI paramagnetic bead-based technology. It enables the purification of high quality RNA from cell lines. The extraction can be run manually in a 2 mL tube format or a 96-well format, or automated in a 96-well format on variety of Beckman Coulter Biomek liquid handling workstations. Total RNA extracted using RNAdvance Cell v2 kit is free of detectable gDNA and other PCR inhibitors.
- Efficient removal of genomic DNA and other contaminants
- Extraction and purification of high quality total RNA from cultured cells supports extraction of a large numbers of samples for gene expression
High Recovery of RNA by RNAdvance Cell v2
RNA was extracted from MRC-5 cells using RNAdvance Cell v2 and other suppliers’ kits. In Figure 1 (below) samples were quantified using the Nanodrop (Thermo Fisher Scientific). Higher amounts of RNA were recovered using the RNAdvance Cell v2 kit over three of the other Suppliers’ kits. In Figure 2 (below) samples were accessed for purity using the Nanodrop (Thermo Fisher Scientific). For MRC-5, RNAdvance Cell v2 purified RNA with A260/280 ratios within satisfactory ratios.
RNA can be Extracted from a Variety of Cell Lines
RNA was extracted from 5x104 MRC-5, ACHN, HCT116 and PANC1 cells using RNAdvance Cell v2. Samples were quantified and accessed for purity using the NanoDrop (Thermo Fisher Scientific). RNA yield is cell line dependent, but RNA quality is consistent between all cell lines and is within satisfactory A260/280 ratios.
|Cell Type||Yield (µg)||A260/A280|
Isolate High Quality RNA with RIN Scores Averaging 9.8 from a Variety of Cell Lines
RNAdvance Cell v2 isolates high quality RNA. Total RNA extracted from MRC-5, ACHN, HCT116, and PANC1 cells and universal human reference RNA (Agilent) were run on the Agilent RNA ScreentTape to assess quality. In Figure 3 (below) RIN values from all samples isolated with RNAdvance Cell v2 were all ≥ 9.4 indicating that high quality and intact RNAs were recovered. In Figure 4 (below) a sample trace of RNA isolated from MRC-5 cells corresponding to the first lane of the gel is shown; the 18s and 28s rRNA peaks are prominent.
Efficient Removal of Genomic DNA and PCR Inhibitors allows for Quality Data and Results in qRT-PCR Applications
The ability to PCR was assessed via qRT-PCR using a primer set (forward primer 5´-ggacttcgagcaagagatgg-3´ and reverse primer 5´-agcactgtgttggcgtacag-3´) designed to span Exon 4 and 5 of the beta (β)- actin gene (ActB) to produce 327 base pair amplicons. In Figure 5 (above) the no RT control demonstrates the removal of DNA that can interfere with downstream RNA applications. In Figure 6 (above) the RNA isolated from 4 cell lines using the RNAdvance Cell v2 kit was amplifiable indicating that the kit removed PCR inhibitors. In Figure 7 (right) RNA extracted from ACHN cells using RNAdvance Cell v2 and 3 other suppliers was used in qRT-PCR with and without reverse transcriptase added. RNAdvance Cell v2 and suppliers A and B had sufficient removal of DNA, but supplier C had equal amplification without reverse transcriptase as with reverse transcriptase indicating DNA contamination.
- Lyse cells in Lysis Buffer and Proteinase K
- Bind RNA to magnetic beads
- Separate magnetic beads from contaminants
- Wash magnetic beads with Wash Buffer and 70% ethanol to remove contaminants
- Treat samples with DNase I
- Rebind RNA to magnetic beads with Wash Buffer
- Wash magnetic beads with 70% ethanol to remove contaminants
- Elute RNA from magnetic beads
- Transfer to a new plate for storage
Extract RNA from Samples in Less Time with Less Pipette Actions Compared to Users of Column-Based Kits
Figure 8 (above) represents total time to extract RNA for 1 to 96 samples using RNAdvance Cell v2 or a column based supplier. At 30 samples total time to extract RNA from cells is faster using RNAdvance Cell v2. In Figure 9 (above) the total number of pipette actions, which include dispensing in a sample, mixing a sample, and discarding tips, required for 1, 8, 24, 48, and 96 samples. With the ability to use a multichannel pipette there is significantly less pipette actions that need to take place than with column based suppliers.
For use in Manual or Automated Methods Based on Batch Size or Overall Throughput
Estimated hands-on time and total time in hours, required to perform 8, 24, 48 and 96 RNA extractions The methods can be performed either manually or automated on a Biomek i7 Hybrid. Difference in time between manual and automation is indicated (NR=not recommended).
|RNAdvance Cell v2||Manual||Automated|
RNAdvance Cell v2 Products for RNA Isolation from Cells
RNAdvance Cell v2 is available in two kit sizes based on your throughput needs. Click on the specific part number below for purchasing or request a quote. For more information check out our RNA isolation from cells page featuring RNAdvance Cell v2.
|A47942||RNAdvance Cell v2 Kit||100|
|A47943||RNAdvance Cell v2 Kit||960|
Not intended or validated for use in the diagnosis of disease or other conditions.
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