Isolate both large and small RNA efficiently
Enrich for small RNA (10-200 nt)
Purify RNA quickly (~30 minutes)
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
The mirVana miRNA Isolation Kits, which employ organic extraction followed by purification on a GFF, use specialized binding and wash solutions that are optimized to prevent loss of the small RNA fraction. The fast and efficient GFF-based method isolates total RNA ranging in size from kilobases down to 10-mers. Figure 1 demonstrates that RNA isolated by the mirVana miRNA Isolation Kit or the mirVana PARIS Kit retains the small RNA fraction.
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 g) 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 -80C.
You Can Also Enrich the Small RNA Fraction
The mirVana Isolation Kit also provides reagents and a procedure to enrich the population of RNAs that are 200 bases and smaller. Small RNA enrichment results in lower background and enhanced sensitivity of small RNA detection by solution hybridization, Northern analysis, and other methods, as compared to the same assay using total RNA. A representative enrichment/depletion example with the mirVana PARIS Kit is presented in Figure 2. The depleted and enriched fractions, or total RNA, were purified from the same number of HeLa cells. Analysis of each fraction on a denaturing agarose gel, a denaturing acrylamide gel or the Agilent 2100 bioanalyzer showed that 28S, 18S, and 5.8 rRNAs were quantitatively recovered in the fraction depleted in small RNA. In contrast, RNAs smaller than 200 nt such as 5S RNA and tRNA were significantly enriched in the complementary "enriched" fraction when compared to the total RNA sample.
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 g 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
In addition to efficient isolation of RNA of all sizes and the optional small RNA enrichment procedure, the mirVana PARIS Kit was also designed to quantitatively recover total proteins from the same sample. This is especially important in experiments where the expression level of both the active miRNA or siRNA and the corresponding target protein need to be measured. Figure 3 demonstrates the effectiveness of the mirVana PARIS Kit for preparing samples for this type of experiment. Here, GAPDH knockdown was triggered by electroporation of a GAPDH-specific siRNA in a primary human cell line (normal human umbilical vein endothelial cells or HUVEC). A reduction of GAPDH expression was observed both at the mRNA and protein levels in these cells, but not in HUVEC cells electroporated with a control Negative Control #1 siRNA sequence (NC). No variation of expression levels was detected for control mRNA (-actin), small RNA (miR-16), and protein (Ku p70).
Figure 3. Using the mirVana PARIS Kit to Analyze RNAi. HUVEC cells (1.5x106) were electroporated with 10 g 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 g) 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 g of total protein and antibodies specific for GAPDH or Ku p70 proteins (Ambion).
The mirVana Isolation Kits include sufficient reagents to perform 40 total RNA isolations or 20 enrichment/depletion procedures. Each purification can accommodate 100-107 cells or 1-100 mg of tissue for the mirVana PARIS Kit, up to 1 g of tissue for the mirVana miRNA Isolation Kit.
Lesslie Beauchamp, Dmitriy Ovcharenko, Emmanuel Labourier Ambion, Inc.