- Extraction and purification of high quality total RNA from many tissue types
- Efficient removal of genomic DNA and other contaminants
- No centrifugation or vacuum filtration required
- No organic extraction steps
- Supports automated as well as manual processing
- Ability to process from eight to 96 samples on a Biomek NX Workstation with a Span-8 configuration
Application: Total RNA Extraction From Tissue
Downstream Application Techniques: qRT-PCR, Microarray Analysis
Manual Process: A. Add tissue to lysis buffer B. Mechanically homogenize and PK digestion.
Automated or Continued Manual Process: 1. Add Binding Buffer 2. Separate magnetic beads from supernatant,
wash with Ethanol 3. DNase I (Optional) 4. Add Rebinding Buffer (Only required when optional DNase
step is performed) 5. Separate magnetic beads from supernatant, wash with Ethanol 6. Elute
Isolating total RNA using the Agencourt RNAdvance Tissue protocol results in high yields without the necessity of centrifugation, vacuum filtration or organic solvents. Isolation of RNA from 10 mg of rat liver using the Agencourt RNAdvance Tissue process resulted in greater than three-fold increase in yield over RNeasy* and greater than two-fold increase in yield over MagMax* (Figure 1). As shown in Figure 2, the Agencourt RNAdvance Tissue system produced a lower Ct value indicating an increased starting quantity of RNA.
|Figure 1 Figure 2
|Figure 1. The Agencourt RNAdvance Tissue system was compared to competitor kits by extracting 10 mg of rat liver tissue in a multi-well format, N = 24 for each kit.
Figure 2. Equal amounts of rat liver was processed using Agencourt RNAdvance and a competitor RNA extraction kit for 8 replicates each. The RT reaction was performed using the Invitrogen first strand synthesis kit using an equal amount from each kit. The qPCR reaction was performed using Applied Biosystems TaqMan Universal PCR Master Mix* on an ABI Prism* 7900. The primers and probe were designed to amplify the rat beta-actin gene.
Variety of Tissue Types
The ability to isolate total RNA can vary greatly between tissue types due to many factors including differences in endogenous levels of RNases and the fibrous or lipid-rich nature of certain tissues. The Agencourt RNAdvance Tissue procedure has been shown to work well with a wide variety of tissue samples. Figure 3 shows the yield of RNA obtained from extraction of five different tissue types including fibrous and fatty tissues.
Figure 3. Total RNA was extracted from several different rat tissues sing the Agencourt RNAdvance Tissue system on a Biomek NX Span-8. Yields are reported as µg/mg of tissue.
N = 24 for liver tissue, N = 8 for all other tissues.
Quality RNA Purification
RNA quality can be readily determined by electrophoresis on an Agilent* Bioanalyzer 2100. Intact, high-quality RNA will have well-defined 28s and 18s peaks with the absence of peak shoulders or an elevated baseline, which are both indicative of degradation. The Bioanalyzer software can calculate an RNA Integrity Number (RIN) based upon these and other qualities in the electropherogram. As seen in Figure 4, RNA extracted using the Agencourt RNAdvance Tissue system results in high RIN scores.
Figure 4. Agilent Bioanalyzer traces of RNA prepared from several
tissues using the Agencourt RNAdvance Tissue system
No Genomic DNA
To test for the presence of genomic DNA contamination, total RNA obtained from the Agencourt RNAdvance Tissue protocol was split into two reactions: one with reverse transcriptase and one without. The resulting samples were then put through PCR for the beta actin gene. The absence of an amplification product in the RT negative control (Figure 5) confirms that genomic DNA was not present in the total RNA.
Figure 5. One (1) µL of total RNA isolated from 10 mg rat liver was used as template for PCR. A 10 µl sample of the reactions were electrophoresed on 1.2% agarose gels. Top gel image: lanes 1-10: RT positive reactions. Lane 11: no template control. Bottom gel image: lanes 1-10: RT negative reactions. Lane 11: no template control. (PCR: amplicon is a 400bp region of the beta actin gene, 35 cycles)