Not All RNA Isolation Procedures Purify Small RNAs
Ambion scientists have developed a line of mirVana™ RNA Isolation Kits for the preparation of miRNA and other small RNA species. Other RNA purification procedures rely on organic extraction followed by alcohol precipitation or adsorption of nucleic acids on a silicate matrix, such as a glass fiber filter (GFF). Unfortunately, alcohol precipitation is time consuming and sometimes inefficient at recovering small RNAs. Furthermore, adsorption to solid supports (e.g. RNeasy® Kit from Qiagen) usually results in loss of most of the small RNA fraction, including small ribosomal RNA (5S rRNA), transfer RNA (tRNA), microRNA (miRNA), or small interfering RNA (siRNA).
Efficient Recovery of Small RNAs
Figure 1. Differential Recovery of Small RNAs. Total RNA was isolated from 1x106 HeLa cells with the indicated kit as per protocol. Total RNA (1 µg) was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide or analyzed by solution hybridization assay with the mirVana™ miRNA Detection Kit (Ambion) and a probe specific for miR-16 prepared by in vitro transcription with the mirVana Probe Construction Kit (Ambion). The gel was exposed for 6 h at -80°C.
You Can Also Enrich the Small RNA Fraction
Figure 2. Small RNA Enrichment Procedure. Total RNA or fractions depleted or enriched in small RNA were isolated from 1x106 HeLa cells with the mirVana™ PARIS™ Kit as per protocol. A 1 µg aliquot of each fraction was resolved on a 1.2% denaturing glyoxal agarose gel or a 15% denaturing acrylamide gel and stained with ethidium bromide. The same fractions were also analyzed with an RNA 6000 NanoChip on the Agilent® 2100 bioanalyzer.
One Sample --> mRNA +Small RNA + Protein
Figure 3. Using the mirVana™ PARIS™ Kit to Analyze RNAi. HUVEC cells (1.5x106) were electroporated with 10 µg of an siRNA targeting GAPDH mRNA or the Silencer® Negative Control #1 siRNA (NC, Ambion) in 400 ml of siRNA Electroporation Buffer (Ambion). Total RNA and protein were isolated with the mirVana PARIS Kit 48 hours after electoporation. Total RNA (1 µg) was used to detect the indicated RNA species by Northern blot or by solution hybridization assay with the mirVana miRNA Detection Kit (Ambion). RNA probes were prepared by in vitro transcription (mRNA probes: MAXIscript® Kit, Ambion) or by 5' end labeling (miRNA and siRNA probes: mirVana Probe & Marker Kit, Ambion). Western blots were performed with 15 µg of total protein and antibodies specific for GAPDH or Ku p70 proteins (Ambion).
Lesslie Beauchamp, Dmitriy Ovcharenko, Emmanuel Labourier • Ambion, Inc.