Navigation Links
Evaluation of RNA isolated from human tissues: quality control for surgical and postmortem samples

James F. Eliason, Ph.D., Asterand plc


Introduction
The path to identification of new and improved drugs for the treatment of human diseases often begins with the identification of novel gene targets through RNA expression analysis. Most high throughput gene expression profiling studies use high density microarrays, which require RNA preparations with purity and integrity of the highest quality. Additional methods for target identification and validation also exist, which may not require as intact RNA. This is especially valuable for gene expression studies in human tissues, as optimal conditions for the preservation of RNA integrity cannot always be met when the samples are excised surgically or from post mortem donors. Thus it is important to have reliable methods for analyzing RNA extracted from human tissues and to understand the level of RNA integrity in each sample, so that the appropriate experimental design may be employed for target evaluation.

RNA purity is generally determined spectrophotometrically. The ratio of absorbances at 260 nm and 280 nm (A260:A280) determines the degree of protein contamination and the A260:A230 ratio is used to identify any contamination by organic solvents. These ratios should be >1.8.

RNA is rapidly digested by RNase enzymes that are nearly ubiquitous, leading to formation of shorter fragments, which can confound experimental results. Thus, it is important to test the RNA integrity. This has been traditionally performed by agarose gel electrophoresis and staining the RNA either with ethidium bromide or SYBR Green dye. In the past, the ratio between the ribosomal bands (28S:18S) was viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of high quality intact RNA. Recent widespread use of the Agilent 2100 Bioanalyzer has shown that this measure is in fact a very poor indicator of RNA quality.

The Bioanalyzer provides a rapid evaluation of RNA with small quantities of material. The electropherogram generated by the instrument provides information in addition to the ratio between the ribosomal bands (see Figure 1). These include:

A) The total area under the 28S and 18S peaks combined

B) The run time for the 18S and 28S peaks

C) The presence or absence of additional peaks between the 28S and 18S peaks or between the 18S peak and the lower marker peak

Using these criteria, Asterand developed a 5 point grading system (Fig. 1), which is very similar to one developed independently by Imbeaud et al. (1). In our system, the grade of 5 indicates the highest possible quality RNA, and 1 indicates a completely degraded sample, unsuitable for rigorous RNA-based experiments. Samples rated grade 3 and above were considered to pass our RNA Quality Control evaluation.


Figure 1. Typical profiles from the Agilent Bio-analyzer 2100 representing different Asterand RNA grades.


The Agilent software now contains an algorithm to calculate an RNA Integrity Number (RIN) where a value of 10 represents the highest quality RNA (2). We have tested this algorithm in comparison to our original method and discovered that the two have a high degree of concordance. Asterand has adapted the RIN measure for our quality control process because of its ease of use, objective nature and automatic generation. Based on comparisons with our original RNA grading system, we can separate the RIN values into 3 categories.

RNA samples with RIN between 7 and 10 are the highest quality and can be used for all types of experiments (Table 1). Those with RIN values between 5 and 7 can be used for many types of experiments, such as PCR, and samples with RIN values below 5 can still be used for some gene expression profiling techniques if they are designed specifically for samples with degraded RNA. These include PCR using short amplicons of 100 bp or less or the Illumina DASL array system.


Table 1.
RIN numbers by application

RIN Suitability 1-5 PCR assays with short regions of amplification 5.1-6.9 qRT-PCR applications 7.0-10.0 Highly demanding gene array assays


The use of alternative technologies can be important when working with human tissue samples. Human tissues often have degraded RNA at the time of collection. This is in contrast to RNA obtained from tissues collected from young, healthy animals or from cultured cells where it is reasonable to demand that all RNA samples have high integrity with RIN values greater than 8.


Effect of Sample Source on RNA quality
Because Asterand assesses the quality of all frozen tissue samples in our XpressBANK, we have a large database of RNA quality results. We have performed an analysis of the RIN values for 28,544 of these fresh frozen samples, and the data are summarized in Table 2. The samples were from both surgical recoveries, primarily cancer, and postmortem recoveries for normal tissues and chronic diseases that are not usually treated by surgery. Approximately 62% of the surgical samples have RIN values greater than 7; whereas only 32% of the postmortem samples have similar quality. The decreased RNA quality of the postmortem samples is related to the cold ischemia time, and also seems to be related to the cause of death and thus length of the agonal period.


Table 2. Distribution of RIN values by source of samples

RIN Category Total Number (%) Postmortem Number ( %) Surgical Number (%) Outstanding RIN ≥7 14922 (53.2) 2859 (31.8) 12063 (61.7) Excellent 5 ≥ RIN <7 4717 (16.5) 2074 (23.1) 2643 (13.5) Good RIN <5 8905 (31.2) 4059 (45.1) 4846 (24.8) Total 28544 8992 19552


These results indicate that in order to have 6 samples with high quality RNA from surgical recoveries, one must collect 10 cases. Similarly, 20 cases must be collected from postmortem recoveries to get the same number of the highest quality samples. Clearly, if large numbers of cases are required for a particular study, then careful selection of assay techniques that allow use of samples with a broad range of RIN values will maximize the ease with which sufficient samples can be found to complete the study. Asterand provides frozen tissue samples with a RIN value for the RNA isolated from a portion of the specimen. In this way, we provide our customers with the most information for making informed decisions about experimental methods to employ.


Asterand Advantage
Asterand provides human tissue samples, RNA, DNA, serum, plasma, fresh blood, cell lines, human primary cells and tissue microarrays. We also provide fresh tissue procurement services. The RNA quality of all the samples in Asterands XpressBANK has been tested, so it is possible to select samples based on this parameter in addition to the clinical and histopathology data associated with them. In this way, the appropriate samples and technologies can be matched for studies.


Asterand Human RNA samples

  • Representative of a broad range of human diseases and conditions
  • Quality assured using Agilent 2100 Bioanalyzer
  • Provided with RIN score and Agilent electropherogram
  • Ethically collected human tissue samples
  • Each RNA sample represents individual donor
  • Associated patient clinical data provided
  • Available ONLINE at http://solutions.asterand.com


References
1. Imbeaud, S, Graudens, E, Boulanger, V , et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res, 2005; 33: e56.

2. Schroeder, A, Mueller, O, Stocker, S , et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol, 2006; 7: 3.



'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Evaluation of FlashPlate in a Helicase Assay
2. Accurate Evaluation of the Finnigan Surveyor Autosampler Performance using a Deferred Standard
3. Practical Aspects of Evaluation of Chromatographic Data in Size Exclusion Chromatography
4. Advancing the quality control methodology to assess isolated total RNA and generated fragmented cRNA
5. Flow cytometric analysis of human primary cells using the Agilent 2100 bioanalyzer and on-chip staining
6. Development of a Multiplex Bead-Based Assay for Antibody Screening of a Nonhuman Primate Colony on the Bio-Plex System
7. Using the Agilent 2100 bioanalyzer for quality control of protein samples prior to MS-analysis
8. Is Z factor the best assessment for the quality of cellular assays delivering higher content?
9. Quality control of antibodies using the 2100 bioanalyzer and the Protein 200 Plus assay
10. Quality assurance of RNA derived from laser microdissected tissue samples obtained by the PALM MicroBeam System using the RNA 6000 Pico LabChip kit.
11. Analysis of urine and seawater samples by ultrasonic nebulization with a high resolution ICP spectrometer
Post Your Comments:
(Date:8/20/2014)... Aug. 20, 2014 /PRNewswire-iReach/ -- A case study ... between the school,s bioengineering department and the Intel® ... Intel® Software Academic Program, UCSD,s research focuses on ... human body.  Photo - ... the work of Dr. Todd P. Coleman ...
(Date:8/19/2014)... 19, 2014 Shimadzu Scientific Instruments ... Prominence-i and Nexera-i, adding to the company’s extensive ... functionality, an intuitive operating environment, and full automation, ... efficient workflow for conventional to ultra-high-speed analysis. ... and intelligent design so users can begin building ...
(Date:8/19/2014)... Robin Williams’ passing is a sad ... an individual. Symptoms range from slowness of voluntary ... and severe depression. Parkinson’s disease progressively gets worse ... producing neurons of the brain. As these patients ... and progressively gets worse, they look for options. ...
(Date:8/19/2014)... 2014   Synthetic Biologics, Inc. (NYSE ... biologic and drug programs targeting specific pathogens that ... its novel C. difficile development program ... Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), ... Synthetic Biologics, Senior Vice President, ...
Breaking Biology Technology:Intel and University of San Diego Bioengineering Department Release Case Study on Health Sciences Research 2Shimadzu’s New i-Series Integrated Liquid Chromatography Systems Provide Laboratories Wider Range of Analytical Capabilities 2Shimadzu’s New i-Series Integrated Liquid Chromatography Systems Provide Laboratories Wider Range of Analytical Capabilities 3As We Mourn Robin Williams’ Passing, His Death Sheds Light on Patients Struggling with Parkinson’s Disease 2As We Mourn Robin Williams’ Passing, His Death Sheds Light on Patients Struggling with Parkinson’s Disease 3Synthetic Biologics Announces Late-Breaking Poster Presentation for C. difficile Program at 54th ICAAC 2Synthetic Biologics Announces Late-Breaking Poster Presentation for C. difficile Program at 54th ICAAC 3Synthetic Biologics Announces Late-Breaking Poster Presentation for C. difficile Program at 54th ICAAC 4
... Kathleen A. Maguire-Zeiss, Brandon K. Harv e y, Rita E. Giuliano and,H ... , Introduction , ... number of factors, including , method ... transfection of mammalian cells has proven to yield ...
... Menu B. Leddy, Biotechnology Research Department,Orange County ... , Introduction ... of bacterial species from membrane , ... of bacterial cells from membrane biofilms cannot be ...
... , Introduction , ... use as primers , or probes in genetic ... traditionally been done by slab gel electrophoresis or HPLC, but these , ... not provide desired quantitative precision or automation. Capillary ...
Cached Biology Technology:Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities 2Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities 3Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities 4Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities 5Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities 6Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 2Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 3Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 4Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 5Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 6Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 7Oligonucleotide Purity Analysis by Capillary Electrophoresis 2Oligonucleotide Purity Analysis by Capillary Electrophoresis 3
(Date:8/20/2014)... screening outcomes of approximately 3 million infants, a team ... the University of Massachusetts Medical School, have shown that ... successfully implemented across public health newborn screening programs. Data ... 20 issue of the Journal of the American ... of SCID in newborns is higher than previously thought ...
(Date:8/20/2014)... that acral melanomas the rare type of skin cancer ... distinct from other more common types of skin cancer, according ... Pigment Cell & Melanoma Research . , Acral melanoma ... the feet, nail-beds and other hairless parts of the skin. ... by UV damage from the sun. , The team, from ...
(Date:8/20/2014)... into a chili pepper causes a burning spiciness that ... exploring the chili pepper,s effect are using their findings ... of pain, which can be caused by inflammation or ... which is being tested in clinical trials, in ACS, ... and colleagues explain that decades ago, scientists had pegged ...
Breaking Biology News(10 mins):Newborn screening expansion offers early diagnosis and treatment to infants with SCID 2Newborn screening expansion offers early diagnosis and treatment to infants with SCID 3Scientists learn more about rare skin cancer that killed Bob Marley 2
... after the world,s first ultrasonic detection device invented ... Virginia Tech Carilion Research Institute scientists have ... have long suspected: ultrasound applied to the periphery, such ... to the brain. And that,s just the ...
... ship is at anchor for longer periods algae, shells and ... economic losses of billions of Dollar. Biological growth on the ... the hull below the waterline which has a braking effect ... basified bio layer, the consumption of fuel can increase by ...
... a simple "drag-and-drop" computer interface and DNA self-assembly ... drug development that could drastically reduce the time ... work supported by a National Science Foundation (NSF) ... NanoLabs of Reston, Va., recently developed and began ...
Cached Biology News:Neuroscientists prove ultrasound can be tweaked to stimulate different sensations 2Neuroscientists prove ultrasound can be tweaked to stimulate different sensations 3Keeping ship hulls free of marine organisms 2Drag-and-drop DNA 2Drag-and-drop DNA 3Drag-and-drop DNA 4
... This DuoSet ELISA Development kit contains the ... sandwich ELISAs to measure natural and recombinant ... and serum. Each kit contains sufficient materials ... plates, provided that the following conditions are ...
Blimp-1 (H-150)...
Mouse Galectin-1 Affinity Purified Polyclonal Ab...
PSGL-1 (215)...
Biology Products: