For phenol-free total RNA extraction from cells/tissues with removal of genomic DNA contamination
Efficient gDNA removal with unique gDNA Eliminator columns (no need for DNase)
High-quality total RNA in minutes using fast and simple extraction protocols
Phenol-free RNA isolation
Ideal for sensitive applications
such as real-time RT-PCR and RNA-Seq
The RNeasy Plus Kits are next generation QIAGEN RNA extraction kits, combining high-quality RNA purification with effective on-column gDNA removal. Total RNA of high purity and yield is obtained from a wide range of cultured cells and easy-to-lyse tissues. The RNeasy Plus Mini Kit purifies up to 100 µg total RNA (>200 nt) from a single extraction with efficient gDNA removal. The RNeasy Plus Micro Kit is specially designed for limited amounts of samples and isolates up to 45 µg pure total RNA. The kits can be automated on the QIAcube and samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent (available separately).
For 250 minipreps: RNeasy Mini Spin Columns, gDNA Eliminator Spin Columns, Collection Tubes, RNase-Free Water and Buffers
The RNeasy Plus Micro and Mini Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Total RNA extraction: high, reproducible yields.
Total RNA was purified in duplicate from different amounts of Jurkat cells using the RNeasy Plus Mini Kit or a similar kit from Supplier AV. Real-time RT-PCR assays for β-actin were performed (40 cycles). The lower CT values with the RNeasy Kit demonstrate greater RNA yields. With the kit from Supplier AV, no transcript was detectable in RNA purified from 102 cells.
Total RNA extraction: effective tissue genomic DNA removal.
Total RNA was purified in duplicate from various mouse tissues (10 mg per sample) using the RNeasy Plus Mini Kit or kits from other suppliers. Real-time PCR assays for c-jun were performed to determine the amount of DNA contamination in the purified RNA.
Total RNA extraction protocol: RNeasy Plus procedure.
A short workflow enables RNA purification with genomic DNA removal in less than 25 minutes.
Total RNA extraction: effective cell genomic DNA removal.
Total RNA was purified from Jurkat cell samples (1 x 106 cells per sample) using [A] the RNeasy Plus Mini Kit, or [B] an RNA purification kit with integrated genomic DNA removal from Supplier AV. Duplicate real-time RT-PCR assays for β-actin were performed with (+RT) or without (-RT) reverse transcriptase. The -RT curves demonstrate that RNA purified using the RNeasy Plus Mini Kit was virtually free of genomic DNA.
Total RNA extraction: array-ready RNA.
Total RNA was purified in duplicate from 1 x 106 HeLa cells using the RNeasy Plus Mini Kit. cRNA was prepared from the duplicate RNA samples (3.5 μg each) using the GeneChip IVT Labeling Kit. The cRNA samples (15 μg each) were analyzed on GeneChip Human Genome U133A probe arrays. The scatter plot shows the correlation between the two samples (Pearson correlation coefficient [r] is 0.996). Red: gene present in both samples; Blue: gene absent or marginal in one sample; Yellow: gene absent or marginal in both samples.
These RNA isolation kits also provide an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. For isolating total RNA containing small RNAs (e.g., miRNA) from cultured cells, additional RNA isolation protocols are available and described in the RNeasy Plus Micro Handbook and as a separate Supplementary Protocol to the RNeasy Plus Mini Kit.
Principle
Cells and easy-to-lyse tissues are first lysed and homogenized in highly denaturing guanidine-isothiocyanate–containing Buffer RLT Plus, which immediately inactivates RNases to ensure isolation of intact RNA. The lysate is then passed through a gDNA Eliminator spin column that, in combination with the high-salt buffer, selectively and efficiently removes genomic DNA. Ethanol is added to provide appropriate binding conditions for RNA, and the sample is applied to an RNeasy spin column. These specialized columns contain a silica membrane that specifically binds RNA from lysed cells.
For fatty and difficult-to-lyse tissues, the RNeasy Plus Universal Kits are recommended. For high-throughput total RNA purification, the RNeasy Plus 96 Kit is recommended. For isolation of total RNA including miRNA from other sample types, we recommend miRNeasy Kits.
Procedure
The RNeasy Plus Micro Kit purifies total RNA from up to 5 x 105 cells or 5 mg tissue, and the RNeasy Plus Mini Kit isolates total RNA from up to107 cells or 30 mg tissue. A short workflow enables RNA isolation with genomic DNA removal in less than 25 minutes (see flowchart RNeasy Plus procedure). Samples are first lysed and homogenized. The lysate is passed through a gDNA Eliminator spin column, ethanol is added to the flow-through, and the sample is applied to an RNeasy spin column. RNA binds to the membrane and contaminants are washed away. High-quality RNA is eluted in as little as 14 µl water using the RNeasy Plus Micro Kit or 30 µl water using the RNeasy Plus Mini Kit.
Different RNA extraction protocols are available for different starting materials. They differ mainly in the lysis and homogenization of the sample. Once the sample is applied to the gDNA Eliminator spin column, the protocols are similar.
When disrupting and homogenizing tissues in Buffer RLT Plus (supplied with the RNeasy Plus Kits), excessive foaming may occur. This foaming is substantially reduced by adding Reagent DX (supplied separately) to Buffer RLT Plus before starting disruption and homogenization. Reagent DX has been carefully tested with the kits, and has no effect on RNA purity or on downstream applications.
Applications
RNA purified using the RNeasy Plus Kits is ideally suited for downstream applications that are sensitive to low amounts of DNA contamination, such as quantitative real-time RT-PCR and RNA-Seq. The purified RNA can also be used in other applications.
The RNeasy Plus Micro Kit is suitable for small samples, including:
