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.
|Product||Description||Size||Quality Standards||Recommended Applications|
|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
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
The Purist Poly(A) RNA Available
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
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.