PRINCIPLE

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.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

• 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

BINDING CAPACITY

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)

RNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.9 – 2.1

RECOMMENDATIONS AND IMPORTANT NOTES

DNA contamination
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.