Navigation Links
Automating RNA Isolation

Large scale gene expression screening projects require reliable high-throughput methods to isolate high quality RNA from a large number of samples. To meet this growing need, Ambion developed the RNAqueous-96 Kit. This kit, which utilizes an RNA-binding glass-fiber filter, provides high yields of intact RNA in a 96-well plate format. An optional on-the-filter DNase treatment can be performed to ensure removal of genomic DNA for RT-PCR applications. Because the kit is compatible with commonly used 96-well plate vacuum manifolds, the procedure is readily automated for use with high throughput liquid handling systems.

Recently, Ambion, in collaboration with PerkinElmer Life Sciences, developed an automation protocol for the RNAqueous-96 Kit using the Packard MultiPROBE II HT Liquid Handling System. This method reproducibly yields high quality total RNA from both adherent and suspended mammalian cultured cells.

Reproducible Isolation of High Quality RNA
To demonstrate the high quality of RNA that can be obtained using the RNAqueous-96 Kit on a robotic platform, RNA from multiple HeLa S3 and K562 cell samples was isolated using the method described in "RNAqueous-96 Automation Protocol". Briefly, HeLa S3 (adherent) and K562 (suspended) cells were transferred to a deep well 96-well plate at 3.75 x 105 cells/well. HeLa cells were incubated for 2 days prior to RNA isolation, whereas K562 cells were processed immediately. The resulting RNA was assessed for both yield and quality on an Agilent 2100 bioanalyzer. Yields were consistently high (~5 pg per cell for HeLa S3 cells and ~8 pg per cell for K562 cells) and RNA samples were uniformly intact. Furthermore, genomic DNA was effectively removed. An electropherogram demonstrating the high quality of the RNA recovered is shown in Figure 1.

Figure 1.RNA Isolated Using the Automated RNAqueous-96 Protocol. Cultured K562 cells (human leukemia, grown in suspension) were pelleted and resuspended in 1X phosphate buffered saline (PBS), counted and transferred to 48 wells of a deep well 96-well plate at 3.75 x 105 cells/well. Total RNA was isolated using the automated method described at "RNAqueous-96 Automation Protocol", using RNAqueous-96 and a MultiPROBE II HT Liquid Handling System with a Gripper Integration Platform. This RNA sample, which was representative of the 48 samples isolated and analyzed, has a 28S rRNA to 18S rRNA ratio of 1.92, indicating a high level of integrity. This isolation yielded 24 g of RNA (344 ng/l in 70l).

Lack of reproducibility can be an issue with some RNA isolation methods. Real-time RT-PCR was used to assess the reproducibility of the automated RNAqueous-96 procedure. Because real-time RT-PCR is quantitative, reproducible, and can be used to analyze multiple samples simultaneously, it is a good choice for such an analysis. Both an abundant (GAPDH) and a rare target (hTERT: catalytic subunit of human telomerase) were individually analyzed in the previously described HeLa S3 and K562 RNA samples using a TaqMan assay. Results from the analysis of 9 samples from K562 cells are shown in Figure 2. Cycle threshold (Ct) values, which reflect the amount of target RNA present in the sample, were calculated (Figure 3). Although some variation in yield between replicate wells was observed, it is most likely due to several factors unrelated to the isolation method, including variation in the number of cells manually dispensed to each well, and variation in cell growth and turnover during incubation. Importantly, the consistent ratio of hTERT to GAPDH in replicate samples indicates that both high abundance and low abundance mRNAs are reproducibly isolated with the automated RNAqueous-96 protocol.

Figure 2.Real-time RT-PCR Detection of GAPDH and hTERT in Replicate Samples Isolated Using the RNAqueous-96 Automation Protocol. RNA was isolated as described in Figure 1. Samples were analyzed by real-time RT-PCR using primers and a TaqMan probe for GAPDH (A.) or hTERT (B.) in an ABI 7700 Sequence Detector. Data from 9 samples, each indicated by a different color line, are shown here. The average cycle threshold (Ct) values for all of the samples are shown in Figure 3.

Cell Line Target Average Ct Value Standard Deviation
K562 GAPDH 15.86 0.45
hTERT 23.63 0.54
hTERT/GAPDH 1.49 0.03 (CV=1.79%)
HeLA S3 GAPDH 16.18 0.8
hTERT 26 1.01
hTERT/GAPDH 1.61 0.30 (CV=2.1%)

Figure 3. Average Ct Values from Real-time RT-PCR Analysis of 48 Samples.

Although this example used the Packard MultiPROBE II HT Liquid Handling System, the RNAqueous-96 Kit is readily adaptable to other automated platforms. The RNAqueous-96 Kit includes reagents and supplies to perform 192 isolations, from 100 to 2 x 106 cells or 0.1 to 1.5 mg of tissue per sample. Reagents for an optional on-the-filter DNase treatment are included.

MultiPROBE is a registered trademark of Packard BioScience Company. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

Ordering Information Cat# Product Name Size 1920 RNAqueous-96 Kit 192 purifications

Related Articles
RNA Conversions
RNA Isolation: The Basics
Which RNA Isolation Kit to Choose?



Page: All 1 2 3 4 5

Related biology technology :

1. Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood
2. High-Throughput Isolation of Total RNA
3. Simple Isolation of RNA from Tissue and Cultured Cells
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
6. Isolation of Microorganisms
7. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
8. Mouse Tail Genomic DNA Isolation Protocol(1)
9. Genomic DNA Isolation Protocol(1,2,3)
10. Basic Plasmid DNA Isolation Protocol
11. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
Post Your Comments:

(Date:11/24/2015)... IN (PRWEB) , ... November 24, 2015 , ... The ... newest Special Interest Group (SIG), MultiGP, also known as Multirotor Grand Prix, to represent ... the last few years. Many AMA members have embraced this type of racing and ...
(Date:11/24/2015)... 24, 2015 --> ... report released by Transparency Market Research, the global non-invasive ... CAGR of 17.5% during the period between 2014 and ... Global Industry Analysis, Size, Volume, Share, Growth, Trends and ... testing market to reach a valuation of US$2.38 bn ...
(Date:11/24/2015)... 24, 2015 /PRNewswire/ - Aeterna Zentaris Inc. (NASDAQ: ... the remaining 11,000 post-share consolidation (or 1,100,000 pre-share ... "Series B Warrants") subject to the previously disclosed ... 23, 2015, which will result in the issuance ... to the issuance of such shares, there will ...
(Date:11/24/2015)... ... November 24, 2015 , ... In harsh industrial processes, ... for in-line sensors can represent a weak spot where leaking process media is ... retractable sensor housings , which are designed to tolerate extreme process conditions. They ...
Breaking Biology Technology:
(Date:11/12/2015)... Nov. 12, 2015  A golden retriever that stayed ... dystrophy (DMD) has provided a new lead for treating ... the Broad Institute of MIT and Harvard and the ... . Cell, pinpoints a protective ... the disease,s effects. The Boston Children,s lab of ...
(Date:11/11/2015)... Minn. , Nov. 11, 2015   MedNet Solutions ... entire spectrum of clinical research, is pleased to announce that ... in Clinical Trials (PCT) event, to be held November ... be able to view live demonstrations of iMedNet ... and learn how iMedNet has been able to ...
(Date:11/9/2015)... , Nov. 09, 2015 ... the addition of the "Global Law ... their offering. --> ) ... "Global Law Enforcement Biometrics Market 2015-2019" ... Research and Markets ( ) has ...
Breaking Biology News(10 mins):