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RNA Extraction using RNAGEM Tissue
and RNAGEM Tissue PLUS
RNAGEM™ Tissue has been developed for rapid preparation of RNA from mammalian cell culture, laser capture micro-dissections and FACS-prepared cell populations, such as macrophages.
RNAGEM™ Tissue uses a rapid protocol for nucleic acid extraction. It releases both RNA and DNA with excellent linearity across a wide range of cell numbers and is automation-ready, closed-tube and requires no further purification of the RNA for accurate RT-qPCR analysis.
Reduced handling, coupled with the efficiency of template preparation by the protease means that RNAGEM Tissue support extraction applications down to single cells.
Kit components
RNAGEM™ Tissue
- RNAGEM enzyme
- 10 x SILVER buffer
- 10 x TE storage buffer
- QuickStart Guide
RNAGEM™ Tissue PLUS
- RNAGEM enzyme
- 10 x SILVER buffer
- RNAse-free DNAse 1
- 10 x DNAse Buffer
- 10 x TE storage buffer
- QuickStart Guide
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Workflow
RNAGEM uses an optimised mix of reagents to allow for the efficient lysis of cells, the removal of RNAses and the proteolytic treatment of the RNA templates. Extraction is carried out at 75°C without needing a higher temperature heat-inactivation step.
When DNAse is used in the PLUS kit, the temperature is dropped after the extraction step and DNAse is added. 5 minutes at 37°C allows the DNAse to act and a subsequent 5 minutes at 75°C reactivates
RNAGEM and the DNAse is hydrolyses without needing to add EDTA.
When compared with Trizol®, the RNAGEM method is fast, simple and hands-free.
Extraction Method
The following procedure outlines general guidelines. Different sample volumes and samples may require slight method variations as described
Preparation
All manipulations should be performed in a clean room or a PCR hood. Use only certified RNAse-free tubes and reagents and equipment and surfaces in 0.05% hypochlorite bleach. Rinse thoroughly with RNAse-free water.
RNAGEM gives linear yields for 10 to 100,000 cells and is ideal for single-cell work. For low numbers of cells we recommend reducing the extraction volume. The minimum volume possible will depend on evaporation with the equipment used,. The recommended amounts of RNAGEM enzyme to use for different extraction volumes are below.
Extraction Volume |
Cell numbers |
Volume of RNAGEM™
|
| 50 - 100 µl | 50,000 - 100,000 | 1 µl |
| 20 - 50 µl | 5000 - 50,000 | 1 µl |
| 5 - 20 µl | 100 - 5000 | 0.5 µl |
| 1 - 15 µl | 1 - 500 | 0.2 µl |
Always use 1/10th volume of SILVER buffer.
For larger cell numbers, the method can be scaled upwards. At high cell densities, the extract will be viscous due to the presence of high molecular weight DNA. This viscosity can be reduced by DNAse treatment, vortexing or repeatedly pipetting the sample.
Sample handling will vary with different sample types. An outline of some suggested procedures is provided below.
Cells in suspension
1. Centrifuge the suspension at 200 x g for 5 mins.
2. Remove all of the liquid.
3. Resuspend the pellet in RNAGEM™ extraction reagents.
Adherent cells
1. Dislodge cells by preferred method (Trypsin or cell scraper).
2. Centrifuge suspension at 200 x g for 5 mins.
3. Remove all of the liquid (a quick spin on a bench centrifuge can help to gather the last few drops).
4. Resuspend the pellet in RNAGEM™ extraction reagents.
Cells stored in RNAlater™
1. Centrifuge suspension at 3,000 x g for 5 mins.
2. Remove all of the liquid (a quick spin on a bench centrifuge can help to gather the last few drops).
3. Resuspend the pellet in RNAGEM™ extraction reagents.
Cell pellets
Up to 5 x 105 cells can be extracted using the recommended method. Linear extraction efficiency is achieved within the range of <10 cells to 105. Cell pellets can be used directly or can be resuspended in 1X SILVER buffer and an appropriate quantity added to the extraction. If greater numbers of cells are to be extracted, then the method should be scaled proportionately.
FACS and LCM
Cells can be collected directly in the extraction reagent mastermix. We recommend using ZyGEM reagents within one hour of preparation. For longer periods, reagents should be frozen, alternatively samples can be collected in a 50% volume of 1x buffer and the rnaGEM reagent and remaining buffer can be added prior to extraction.
RNAGEM™ is sensitive to EDTA and other chelating agents. If cells are presented in EDTA-containing solutions, they should be centrifuged at 200 x g and washed in 1X SILVER buffer before use.
Extraction
(50 µl reaction described below - see notes above on scaling)
RNAGEM Tissue (no DNAse treatment)
1. Add:
- Cell suspension or pellet
- 5 µl 10x SILVER Buffer
- 1 µl RNAGEM
- Water to a final volume of 50 µl
2. Vortex and incubate:
- 75°C for 5 min (< 50,000 cells) or 10 min (> 50,000 cells)
- 4°C HOLD
- A thermal cycler should be used for this step.
3. Add 1/10th volume of 10 x TE Buffer (provided). Store at -20°C.
RNAGEM Tissue PLUS (DNAse I treatment)
1. Add:
- Cell suspension or pellet
- 5 µl 10x SILVER Buffer
- 1 µl RNAGEM
- Water to a final volume of 50 µl
2. Vortex and incubate:
- 75°C for 5 min (< 50,000 cells) or 10 min (> 50,000 cells)
- 4°C HOLD
- A thermal cycler should be used for this step.
3. Leave in the thermal cycler at 4°C and add:
- 5 µl DNAse buffer
- 2 µl DNAse I
- Scale for different volumes
4. Vortex and incubate in thermal cycler as follows:
- 37°C for 5 min
- 75°C for 5 min
- 4°C HOLD
5. Add 1/10th volume of 10 x TE Buffer (provided). Store at -20°C.
Technical tips and sample management
- The method, the enzyme formulation and buffer have been carefully optimised for extracting intact RNA. Using the enzyme with other methods or buffers is not recommended. If you need to modify the method in any way, please email: support@zygem.com.
- Absorbance 260/280 nm is an ineffective quantification method with RNAGEM-prepared nucleic acids. For accurate quantification we recommend RT-qPCR and normalisation to genomic DNA using a reference gene. Please see our Application Note on RNA quantification. <Z01071_AppNote108_RNAGEMQuant_US.pdf>
- As with any method RNA preparation, the best results are obtained when samples are handled on ice in an RNAse-free environment and using certified RNAse-free tubes and reagents.
- For long-term storage, RNA should be stored at -80°C.
- Alternatively, RNA in TE buffer can be precipitated using NH4OAc/ethanol (0.1 volumes of 5 M NH4OAc, and 2.5 volumes 100% ethanol) and stored at -20°C or below.
Typical Results
The SILVER buffer used by RNAGEM has been formulated to be compataible with polymerases and reverse-transcripases and so the extracts can be used without purification in PCR, RT-PCR, qPCR and RT-qPCR.
The plots below were obtained when 5 µl of RNAGEM extracts were added directly to an RT-qPCR. HeLa cell numbers from 10-50,000 were RNAGEM-treated and plots generated from a high abundance mRNA (ACTB; ß-actin) and a low abundance mRNA (BRCA1; breast cancer early onset). The clean traces with gradients similar to the standards demonstrate the lack of inhibition.
Figure 1: RT-qPCR plots of RNA extracted from a dilution series of HeLa cells from 10 - 50,000 cells. A: high copy number mRNA (ACTB). B: low copy number mRNA (BRCA1). Red = standards; Blue = duplicate mRNA curves
Using the recommended method, RNAGEM extraction efficiency is constant over the range of 1 to approximately 50,000 cells and gives excellent yields (see below). Different ranges of linearity can be obtained by small modifications to the base procedure. Advice on modifying the procedure can be obtained from support@zygem.com.
Figure 2: Log/Log plots of HeLa cell numbers versus mRNA copies detected. Both high copy number mRNAs (ACTB) and low copy number (BRCA1) are shown.