NUCLEIC ACIDS EXTRACTION

Nucarrier

Tri-RNA Reagent

After Tri-Reagent
RNA Clean-Up Kit

miRNA Isolation Kit

RNA Stabilization Solution

Description
The Favorgen miRNA Isolation Kit is designed for purification of microRNAs (miRNAs) and other small cellular RNA s from tissue samples or cultured cells. Purification of miRNAs allows research into biological significant pathways for regulation of gene regulation. The standard protocols for isolating total RNA and mRNA are not optimized for isolation of small RNA molecules and result in the loss of substantial amounts of miRNAs and other small RNA. In addition, removal of the predominant larger RNAs is required for accurate analysis of miRNA expression by qPCR or microarray analysis. This kit is specifically designed for purification of small RNA with minimal contamination from large RNA molecules or genomic DNA. The method employs a spin column with a silica-based fiber matrix that binds RNA in the presence of a chaotropic salt. The method is based on the selective binding of RNA molecules of different sizes to the silica-based fiber matrix when different ethanol concentrations are present in the solvent.

Kit Components
Lysis Buffer-10 ml
Acid-Phenol-10 ml
RNA Column and Collection Tube-100 sets
Wash Buffer-10 ml
Release Buffer-0.5 ml

Required Materials
Ethanol
Isopropanol
Chloroform
Microcentrifuge
Water Bath or Dry Bath or Microwave Oven

miRNA Isolation Protocol from 100 mg tissue or 1xe6 cultured cells
•  Add 200 μl Lysis Buffer into the tube containing 100 mg tissue or 1x 10 6 cultured cell pellet

•  Vigorous mixing by vortexing. Incubate at room temperature for 10 minutes.

•  Add 200 μl Acid-Phenol and 40 μl chloroform into the tube, vortex vigorously for 2 minutes.

•  Centrifuge at 12,000 rpm for 3 minutes. Transfer the upper phase into a clean tube

•  Add ethanol to 35% volume (ex., add 108 μl ethanol to 200 μl upper phase). Mix well.

•  Transfer to the RNA Column in the Collection Tube . Incubate for 1 minute.

•  Centrifuge at 12,000 rpm for 30 seconds. Collect the filtrate.

•  Add ethanol to 70% volume (ex., add 338 μl ethanol to 290 μl upper phase). Mix well.

•  Transfer to another RNA Column in the Collection Tube . Incubate for 1 minute.

•  Centrifuge at 12,000 rpm for 30 seconds (miRNA bound to the column membrane).

•  Add 200 μl Wash Buffer . Incubate for 1 minute.

•  Centrifuge at 12,000 rpm for 1 minute to completely remove the residue liquid.

•  Put the RNA Column to a clean 1.5 ml tube.

•  Add 50 μl Release Buffer (preheated to 65°C ) to the center of column. Incubate for 3 minutes.

•  Centrifuge at 12,000 rpm for 3 minute to recover miRNA. (Note: The purified miRNA can be further concentrated by a standard ethanol precipitation procedure and then re-dissolved in a small volume ddH₂O or TE, pH 8.0)

•  Use 1/5 volume to run on a mini agarose gel (or more accurately, a polyacrylamide gel) to check its quality. The majority of RNA visible on the gel should be <100 nt in size, with the major bands corresponding to tRNAs. The 5S and 5.8S rRNA species may also be visible. These tRNA and small rRNA bands should be clear and distinct. miRNA (21-22 nt) are typically not visible on the gel image.

•  Store the samples at – 20 ℃ and used for cDNA synthesis.