Ambion's mirVana PARIS Kit combines the technological advances from both the mirVana miRNA Isolation Kit, optimized for small RNA recovery, and the PARIS (Protein And RNA Isolation System) Kit, specifically developed to isolate protein and RNA from the same experimental sample (see MicroRNAs: Isolation to Functional Analyses). The result is a versatile kit that provides quantitative recovery of native protein and all RNA species, including siRNA and miRNAs. The kit is ideal for downstream assessment of protein and mRNA expression levels, analysis of microRNA (miRNA) expression patterns, and correlation of siRNA expression with protein and mRNA knockdown in RNAi experiments .
Reproducible, Quantitative Recovery of Protein and RNA
There is a direct relationship between the inefficient recovery of 5S rRNA and the loss of smaller RNA species, such as miRNAs and siRNAs, during total RNA isolation . For example, total RNA samples prepared with an RNA isolation procedure not optimized for small RNA recovery lack RNA species smaller than ~200 nt, including 5S rRNA, tRNA, and miRNA (Figure 1, PARIS lane). Even with the sensitivity of the mirVana miRNA Detection Kit [2, 3], miR-16 miRNA could barely be detected in 1 g of total RNA isolated from HeLa cells in this way. In contrast, RNA isolated using both the mirVana miRNA Isolation Kit and mirVana PARIS Kit procedures included these small RNA species.
Figure 1. Differential Recovery of Small RNAs. Total RNA was isolated from 1x106 HeLa cells with the indicated kit as per protocol. One g of total RNA was resolved on a 15% denaturing acrylamide gel and stained with EtBr (top) 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). Gel was exposed for 6 (middle) or 24 (bottom) hours at -80C
Variation in protein, mRNA, and/or small RNA recovery can be an issue if precise quantitation is required in downstream experiments (e.g. RNAi experiments, miRNA profiling). Therefore, reproducible, quantitative recovery of RNA and protein is critical. To determine the reproducibility of the mirVana PARIS procedure, both RNA and protein were isolated from replicates of two types of biological samples, and various quantitative and qualitative parameters were measured. The experimental samples tested were human cell lines assayed 2 days after plating (12 replicates, Figure 2), and mouse liver tissue kept frozen for 2 months at -80C prior to assay (6 replicates, Figure 3). For each experiment, total RNA and protein yields were determined, and samples were assessed for effi cient recovery of long RNA species (real time RT-PCR), small RNA (gel or Northern blot), and protein (Western blot). The results showed that high quality total RNA and protein were efficiently recovered from both samples types. No apparent degradation was observed by gel analysis or Western blot. The mirVana PARIS isolation procedure was also extremely reproducible as each independent replicate contained the same relative proportion of long RNA (18S rRNA, GAPDH, and/or Cyclin D1 mRNA), small RNA (5S rRNA), and protein (GAPDH). Quantitative analysis by real time PCR showed variations of less than 10% (Figure 4).
Figure 2. Analysis of 12 Human Cultured Cell Replicates. HeLa cells were plated at 200,000 cells per well in 6 well plates. Total RNA and protein were isolated 2 days after plating with the mirVana PARIS Kit. One g of total RNA was analyzed on a 15% denaturing polyacryl-amide gel stained with EtBr (left top). Ten g of total protein was resolved on a 10% SDS polyacrylamide gel and analyzed by Western blot with an anti-GAPDH antibody (Ambion) (left bottom). Real time PCR reactions were performed in duplicate using the indicated gene specific primers in combination with SYBR green technology (right).
Figure 3. Analysis of 6 Mouse Liver Replicates. Total RNA and protein were isolated with the mirVana PARIS Kit from 50-80 mg pieces of liver dissected from 6 different mice and kept frozen at -80C for 2 months. One g of total RNA was analyzed on a 1.2% denaturing glyoxal agarose gel stained with EtBr or resolved on a 15% denaturing polyacrylamide gel and analyzed by Northern blot with a 5S rRNA pr obe 5' labeled and purified with the mirVana Probe & Marker Kit (Ambion). GAPDH protein levels were analyzed as in Figure 2. Real time PCR reactions were performed in quadruplicate using TaqMan primer and probe sets for 18S rRNA and GAPDH mRNA (Applied Biosystems).
Figure 4. Summary of Real Time PCR Results. Average Ct values, the range of Ct values, and standard deviations are given for the replicate samples used in real time RT-PCR experiments in Figures 2 and 3.
The mirVana PARIS Kit is a robust method ideal for quantification of protein and RNA, including small RNA species. The kit contains sufficient reagent for 40 protein and RNA isolations; each purification can accommodate 100 to 107 cells or 1 to 100 mg of tissue. The entire procedure is fast and easy, isolating RNA and proteins in less than 30 min. Also, the mirVana PARIS Kit is compatible with samples treated with RNAlater (Ambion), a reagent that stabilizes RNA within tissue prior to processing. For more information about the mirVana PARIS Kit and other tools dedicated to small RNA analysis, please visit www.ambion.com/miRNA.
Cat# Product Name Size 1556 mirVana PARIS Kit up to 40 purifications