Isolate siRNA, miRNA, Total RNA, & Native Protein

• Isolate total RNA and native protein from the same sample
• Enrich for small RNAs (<200 nt)
• Ideal for assessing siRNA or miRNA expression and processing

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) have emerged as powerful post-transcriptional regulators of gene expression in many different organisms, thus making the analysis of small RNA molecules increasingly important. In addition to monitoring small RNA expression, analysis of protein expression levels is critical for thorough analysis of the effects of small RNAs. In RNAi experiments, for example, exogenously introduced siRNAs are used to target the degradation of specific messenger RNAs (mRNAs), resulting in gene knockdown at both the mRNA and the protein level. In contrast, miRNAs are endogenous 21-24 nt RNAs that primarily act as repressors of translation and therefore affect only protein expression levels. Here we discuss a procedure for downstream monitoring of protein, mRNA, siRNA, and miRNA expression levels from the same experimental sample.

To isolate protein, small RNA and long RNA species from the same sample, Ambion scientists have developed a unique tool: the mirVana PARIS Kit. Quantitative recovery of small RNA during total RNA isolation requires optimized procedures; Ambion's mirVana miRNA Isolation Kit was specifically optimized for efficient isolation of RNA species smaller than 200 nt [1]. To develop the mirVana PARIS Kit, this technology was adapted and combined with the advantages of the PARIS Kit, the first commercially available kit designed to isolate both RNA and native protein from the same sample [2]. The result is a versatile kit for quantitative recovery of native protein and all RNA species, including small RNAs. A summary of each kit's specification is presented in Figure 1, and specific examples are provided below.

Figure 1. Which Kit to Choose?

To assess the quality of total RNA isolated with the mirVana PARIS Kit, 1 x 10⁶ HeLa cells were homogenized in the Cell Disruption Buffer provided with the kit. Because the homogenization is performed quickly on ice and in the presence of detergent, both protein and RNA can be recovered from the lysate. A fraction of the lysate can be used directly to analyze protein expression (e.g. Western blot, 2D gel electrophoresis) or for assays that require native protein (e.g. functional assay, reporter activity). For RNA isolation, the HeLa cell lysate was immediately mixed with an equal volume of 2X Denaturing Solution. This solution contains a high concentration of chaotropic denaturant, resulting in rapid inactivation of cellular ribonucleases. After a rapid extraction with Acid-Phenol:Chloroform (provided with the kit), total RNA was then purified from the mixture using an RNA-binding glass fiber filter (GFF) and optimized binding and wash buffers (also provided).

In parallel, total RNA was isolated from the same number of cells with the mirVana miRNA Isolation Kit and with the PARIS Kit (the latter is opimized for longer RNA isolation and does not recover small RNAs efficiently). Comparison of the purified material on a denaturing polyacrylamide gel showed equivalent recovery of small RNA with the mirVana and mirVana PARIS Kits, but RNA species smaller than ~200 nt were absent in the sample processed with the PARIS Kit (Figure 2A). Previous studies [1] demonstrated a correlation between loss of 5S rRNA and tRNA, and inefficient recovery of miRNA or siRNA (see also Figure 3 and 6) using standard GFF protocols. The integrity of the isolated total RNA was assessed on an RNA 6000 Nano LabChip with an Agilent 2100 bioanalyzer. As seen in Figure 2B, RNA isolated with the mirVana PARIS Kit was of high quality (28S/18S rRNA ratio 1.77). The presence of small RNA was evidenced by a clear peak at ~26 seconds.

Figure 2. Analysis of Total RNA Isolated with the mirVana PARIS Kit. Total RNA was isolated from 1 x 10 ⁶ HeLa cells with the indicated kit as per protocol. (A) 1 µg of total RNA was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide. (B) Electropherogram of total RNA purified with the mirVana PARIS Kit analyzed on an Agilent 2100 bioanalyzer.

Small RNA analysis often requires the use of extremely large amounts of input RNA. Furthermore, small RNA and mRNA species are analyzed with techniques that are not compatible, for example gel purification or Northern blotting on 15% acrylamide gels for miRNA/siRNA vs. RT-PCR or microarray analysis for mRNA. With the mirVana PARIS Kit, fractions enriched in RNA species smaller than 200 nt and fractions containing only the longer RNA species can be prepared from the same experimental sample using differential binding conditions on GFF. Thus the kit allows for separate downstream analysis of protein, small RNA, and mRNA if required.

Representative data of enrichment/depletion of small RNAs obtained with the mirVana PARIS Kit are presented in Figure 3. The depleted and enriched fractions, or total RNA, were purified from the same number of HeLa cells in triplicate. Analysis of 1 µg RNA on a denaturing agarose or acrylamide gel showed that 28S, 18S and 5.8S rRNA 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 corresponding "enriched" fraction when compared to the total RNA sample. Quantitative analysis of the same samples by Northern blot confirmed that smaller RNA species such as the microRNA miR-16 (22 nt) were also enriched ~10 fold (Figure 3B).

Figure 3. Small RNA Enrichment from Cultured Cells. Total RNA or fractions depleted or enriched in small RNA were isolated in triplicate from 1 x 10 ⁶ HeLa cells with the mirVana PARIS Kit as per protocol. (A) 1 µg of each fraction was resolved on a 1.2% denaturing glyoxal agarose gel and stained with ethidium bromide. (B) 1 µg of each fraction was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide. After electrotransfer, miR-16 miRNA was detected by Northern blot with an antisense RNA probe 5' labeled and purified with the mirVana Probe & Marker Kit (Ambion).

Researchers often need to assess the specificity of gene silencing experiments and to correlate knockdown of a target mRNA with a reduction in the corresponding protein levels. Determining expression levels of active siRNA is also an important parameter that needs to be assessed. Figure 4 demonstrates the effectiveness of the mirVana PARIS Kit for preparing samples for this type of experiment.

Figure 4. Using the mirVana PARIS Kit to Analyze RNAi Effect. 1.5 x 10 ⁶ HUVECs were electroporated (800 V, 120 µs, 2 pulses, 0.5 s between pulses) with 10 µg of siRNA targeting GAPDH mRNA or Silencer Negative Control #1 (Ambion) in 400 µl of siPORT siRNA Electroporation Buffer (Ambion) using 4 mm electroporation cuvettes and square wave type pulses. Total RNA and protein were isolated with the mirVana PARIS Kit 48 hours after electoporation. 1 µg of total RNA 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 (rRNA, 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 (Ambion).

Here, GAPDH knockdown was triggered by electroporation of a GAPDH-specific siRNA into 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 level in these cells, but not in HUVECs electroporated with Silencer Negative Control #1 siRNA. No variation in expression level was detected for the control mRNA (ß-actin), small RNAs (miR-16, 5S rRNA, 5.8S rRNA), or the control protein (Ku).

Although it was specifically designed to quickly process cultured cell samples, the mirVana PARIS Kit can also be used to isolate RNA and protein from most animal tissues. During the rapid homogenization step in Cell Disruption Buffer, RNA can be partially accessible to cellular ribonucleases. Thus, the procedure is not compatible with tissues that contain very high levels of these enzymes, such as pancreas or spleen. To test for potential loss of RNA integrity during sample homogenization, total RNA was isolated from four different mouse tissues with the mirVana PARIS Kit or the mirVana miRNA Isolation Kit. With the latter kit, tissue samples are homogenized directly in a solution containing a chaotropic denaturant, which reduces the opportunities for RNA degradation.

Comparison of the purified RNA on a denaturing gel showed no significant RNA degradation with either kit (Figure 5A). Most importantly, analysis of small RNA expression profiles showed no difference between the two procedures. let-7 microRNA was more abundant in brain than kidney, liver, or thymus, whereas both 5S and 5.8S rRNA were detected at the same level in all 4 tissues. As previously reported [3, 4], miR-124 was expressed only in brain. Together, these results show that homogenization in Cell Disruption Buffer prior to addition of chaotropic denaturant does not significantly affect RNA quality. The advantage is that this lysate contains native protein that can be used directly in a host of downstream applications [2, 5], such as two-dimensional gel electrophoresis (Figure 5B).

Figure 5. Isolation of Total RNA and Protein from Different Tissues. Total RNA and protein were isolated from 4 different mouse tissues (~50 mg) with the mirVana miRNA Isolation Kit (RNA only) or the mirVana PARIS Kit. (A) Small RNA analysis. One µg of total RNA was analyzed by denaturing gel and Northern blot as described in Figures 2 and 3.(B) Two-dimensional gel analysis. About 125 µg of total protein from mouse brain was resolved using a pH 4-7 IPG gel followed by a 8-16% SDS-PAGE and stained with Coomassie blue.

To further examine the compatibility of the mirVana PARIS Kit with tissues, the enrichment procedure was performed with mouse brain or kidney that were stored for several months in RNAlater, Ambion's tissue collection/stabilization solution. Previous studies showed that both high quality protein and RNA are recovered from RNAlater-treated samples [2, 6]. Analysis by denaturing acrylamide gel and Northern blot of the RNA samples purified with the mirVana PARIS Kit confirm that the procedure efficiently fractionates long and short RNA species (Figure 6). mRNAs and 5.8S rRNA were detected only in the "depleted" fraction, and the miRNAs let-7 or miR-124 were seen only in the "enriched" fraction.

Figure 6. Small RNA Enrichment from Mouse Tissues. Total RNA or fractions depleted or enriched in small RNA were isolated from RNA later-treated mouse brain or kidney (~50 mg) with the mirVana PARIS Kit (A) 1 µg of each fraction was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide. (B) mRNA, rRNA and miRNA expression was analyzed as described in Figure 4. This figure demonstrates the broad range of RNAs that can be obtained with this procedure, from miRNAs (~22 nt) to much larger mRNA species (in the kilobase range).