The EXTRACTME miRNA KIT is designed for the rapid and efficient purification of miRNA with possibility of simultaneous purification of large RNA and DNA. High quality miRNA may be purified from 1-10 mg of tissue (fresh or frozen) and 104-106 cultured cells. The isolation protocol, buffers formulations and columns were optimized for high isolation efficiency and purity of miRNA. The product is intended for research use only.
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The EXTRACTME miRNA KIT utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids at high concentration of chaotropic salts. The isolation procedure consists of 7 steps. In the first isolation step, the tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). Then the homogenate is lysed with guanidine thiocyanate and detergents. Any RNases are inactivated by guanidine thiocyanate and ß-mercaptoethanol. In next step DNA is separated by binding to the first minicolumn. Next the large RNA is bound to the Large RNA Purifcation Column membrane by selective conditions in mixture after addition of ethanol. The miRNA is bound in next step to the miRNA Purifcation Column. The three-step washing stage effectively removes impurities and enzyme inhibitors. The purified miRNA is eluted using a low ionic strength buffer or RNase-free water (pH 7.0-9.0) and can be used directly in all downstream applications such as RT-PCR, Northern blotting, RT-qPCR and so forth.
- fresh or frozen tissue (stored at -80°C): 1 – 10 mg
- tissue preserved in RNase inactivating buffers: 1 – 10 mg
- cell culture: 104 – 104 cells
Approx. 90 μg
miRNA TIME REQUIRED
- 25 – 30 minutes (lysis and homogenization time not included)
- 35 – 70 minutes for homogenization in liquid nitrogen
- 35 – 50 minutes for mechanical homogenization (ceramic beads)
A260/A280 ratio = 1.9 – 2.1
RECOMMENDATIONS AND IMPORTANT NOTES
All the biological material used for miRNA isolation also contains DNA. There is no RNA isolation method which guarantees complete DNA removal unless the RNA/miRNA sample is treated with DNase after isolation. Even slight DNA contamination (several gDNA copies per reaction) may give an additional signal in a quantitative PCR analysis after reverse transcription. In order to avoid this, we recommend treating the purifed miRNA sample with an appropriate enzyme (eg. termolabile dsDNase, cat. no. EN32). We also recommend designing primers which are insensitive to DNA contaminations (primers in adjoining exons or with intron >1.5 kbp) for the purposes of qPCR analysis.
EXTRACTME miRNA KIT (EM12) - Manual
EXTRACTME miRNA KIT – TROUBLESHOOTING
The quality of each production batch (LOT) of the EXTRACTME miRNA KIT is tested using standard QC procedures. The purifed RNA concentration, quality and stability are evaluated by gel electrophoresis and spectrophotometer. In addition, the functional quality is tested by reverse transcription and qPCR.
EXTRACTME miRNA KIT (EM12) - MSDS
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