With the emergence of more and more sophisticated techniques for gene expression analysis (e.g. array analysis, real-time RT-PCR), the quality of the RNA used in these studies has become increasingly critical. As The RNA Company®, Ambion is committed to defining what "high quality RNA" truly means and providing the best RNA available from any source. Ambion's newest RNA products include RNA from specific regions of the human brain and other human RNAs from diverse normal and diseased tissues.
RNA Isolation: Leave it to Ambion
Obtaining high quality RNA, especially human RNA, can be challenging because of factors that are often difficult to control. Tissue procurement is probably the biggest hurdle for many sample types. First a reputable and ethical tissue source must be identified. Typically you then wait for the desired tissue type and sample size to become available, and finally you hope that the tissue was harvested quickly and handled properly to avoid RNA degradation. Once you have obtained the desired tissue sample, it must be disrupted and homogenized using the technique and reagents best suited to the tissue source. After completing the RNA isolation procedure, it is also crucial for demanding applications to assess the quantity, quality, and purity of the RNA. For successful gene expression analysis and cloning experiments, accurately quantitated, full length, pure RNA is required. Many applications also demand that the RNA be purified of contaminants such as genomic DNA, ribosomal RNA (in mRNA preps), protein, ethanol, organic solvents, and salt.
Ambion's prepared RNAs are available for researchers who choose to avoid these challenges. Our FirstChoice® RNAs are available from a variety of human, mouse, and rat tissues, and from cell lines. The FirstChoice RNAs, which are stored in THE RNA Storage Solution to promote long term stability, pass the industry's most stringent quality control tests. The chart in Figure 1 describes the different RNA products available and the applications for which they are recommended.
|FirstChoice® Total RNA ||Total RNA from specific human, mouse and rat tissues, human brain regions, human and Drosophila cell lines and E.coli* ||Human Tissues & Cell Line- 100 µg Brain Region- 50 µg Mouse & Rat- 200 µg E.coli- 200 µg ||Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR ||RT-PCR, Array Analysis, Northern Analysis, RPA, cDNA Library Construction
|FirstChoice® Poly(A) RNA ||Poly(A) RNA from specific human, mouse and rat tissues and human and Drosophila cell lines* ||5 µg ||Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR, mRNA enrichment determined by Northern analysis ||RT-PCR, Array Analysis, Northern Analysis, RPA, cDNA Library Construction
|FirstChoice® Tumor/Normal Adjacent Tissue RNA ||Total RNA from pairs of individual human tumor and normal adjacent tissues* ||5 µg of each ||Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR ||RT-PCR, Array Analysis, RPA cDNA Library Construction
|FirstChoice® Tumor RNA ||Total RNA from individual human tumors* ||5 µg ||Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR ||RT-PCR, Array Analysis, RPA cDNA Library Construction
|*All human RNAs include donor demographic information and, when applicable, pathology/histology information.
Figure 1. Description of Ambion's High Quality RNAs.
RNA from Human Brain Regions is Now Available
Ambion now offers high quality FirstChoice Total RNA from individual regions of the human brain. Traditionally, it has been difficult for researchers to obtain high quality RNA from human brain tissue. Ambion has established relationships within the research and medical community to ethically obtain tissues in a manner that preserves the integrity of the RNA. We have also optimized RNA isolation procedures specifically for brain and neural tissue. These efforts, in conjunction with stringent quality control standards, ensure that FirstChoice Brain Total RNA is intact, stable, and free of genomic DNA (<0.05%). Results from one of our Quality Control assays are shown in Figure 2.
Figure 2. FirstChoice™ Human Brain Total RNA Stability
. RNA was stored overnight at either -20ºC or 37ºC. The RNA was then evaluated on an Agilent 2100 bioanalyzer. The approximate 2:1 ratio of the peaks representing 28S and 18S rRNA indicates that the RNA is intact and stable even when stored overnight at 37ºC.
Replicate samples of total brain RNA were stored overnight at either 20ºC or at 37ºC and then analyzed by capillary electrophoresis using the Agilent 2100 bioanalyzer. Even after overnight incubation at 37ºC, the ratio of the areas of the peaks representing 28S and 18S rRNA are approximately 2:1; this illustrates the high quality and stability of FirstChoice RNA. Total RNA is currently available from the following regions of the brain: frontal cortex, occipital cortex, pariental cortex, temporal cortex, basal ganglia, cerebellum, medulla, and thalamus.
Other High Quality Total RNAs
FirstChoice Total RNA is available from a wide variety of human, mouse, and rat tissues and cell lines. The samples are processed using Ambion's RNA isolation reagents to recover highly pure, intact RNA. A stringent DNase treatment is included to destroy any contaminating genomic DNA, and to ensure that the RNA is ready for use in downstream applications such as RT-PCR. Although it is impossible to remove every trace of DNA contamination from an RNA sample, FirstChoice RNAs are tested by real-time PCR to ensure that residual DNA contamination is insignificant (<0.05%). Additionally, the RNAs undergo rigorous testing for nucleases. The integrity and stability of each RNA is verified by capillary electrophoresis using the Agilent 2100 bioanalyzer as shown in Figure 2.
The Purist Poly(A) RNA Available
FirstChoice Poly(A) RNA is isolated from the FirstChoice Total RNA described above. The RNA undergoes poly(A) selection using Ambion's Poly(A)Purist™ technology
; a method that outperforms other purification methods and yields RNA that is more highly enriched for poly(A) RNA and is nearly devoid of rRNA. Because of the high quality of starting material (FirstChoice Total RNA), and the unsurpassed level of mRNA enrichment, 5 µg of Ambion's FirstChoice Poly(A) RNA contains more mRNA than other commercially available poly(A) RNA preparations which are often highly contaminated with rRNA, DNA or both. An electropherogram of FirstChoice Poly(A) RNA is shown in Figure 4; it illustrates the purity of the preparation.
Figure 4. FirstChoice™ Human Prostate Poly(A) RNA Quality.
Poly(A) RNA (100 ng) was evaluated on an Agilent 2100 bioanalyzer. The resulting electropherogram shows intact mRNA that is free of residual ribosomal RNA.
Tumor and Tumor/Normal Adjacent Tissue RNA Pairs
Also part of the FirstChoice line of products, the FirstChoice Tumor
and FirstChoice Tumor/Normal Adjacent Tissue total RNAs are prepared using the same methods and quality standards as the FirstChoice Total RNAs, but are derived from human tumor samples. The FirstChoice Tumor RNAs include 5 µg of total RNA from an individual human tumor. The FirstChoice Tumor/Normal Adjacent Tissue RNA pairs include 5 µg each of total RNA isolated from an individual tumor and the normal adjacent tissue (NAT).
All human tissues used for RNA isolation at Ambion are obtained through accredited organ/tissue procurement organizations that operate with Institutional Review Board approval, and in accordance with regulations set forth by the United States Department of Health and Human Services. These regulations include donor consent and protection of privacy regulations.