tRNA


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. This leads to far greater sensitivity and linearity across a broad range of cell numbers.

 

The plots show below were obtained when 5 µl of RNAGEM extracts from HeLa cells were added directly to an RT-qPCR. Cell numbers ranging from 10 - 50,000 were RNAGEM-extracted 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 and the plots obtained from as few as 10 cells demonstrate the sensitivity of the method.

 

RNAGEM qPCR

 

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, the efficiency of RNAGEM extraction is constant over the range of 1 to approximately 50,000 cells and gives excellent yields (see below). In contrast, Trizol extraction loses the mRNA from low abundance cells. This means that sample normalisation can be obtained with a simple qPCR of gDNA in non-DNAse-treated extract. See our Application note for more details. 

 

Different ranges of linearity can be obtained by small modifications to the base procedure (as shown in the table on the first column).

 

RNA linearity 

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.

 

 

Technical tips and sample management

 

1. 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 let us contact us through our technical support.

 

2. 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.

 

3. 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.

 

4. 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.


Downloadable PDF files

 

RNA extraction using RNAGEM (PDF 876k)

Quant/normalisation of RNA yields with RNAGEM (PDF 217k)

RNAGEM Tissue Quickstart Guide (PDF 171k)

RNAGEM Tissue PLUS Quickstart Guide (PDF 175k)

RNAGEM MSDS (PDF 179k)


Extraction method 

 

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.

 

RNAGEM Tissue and RNAGEM Tissue PLUS have been developed for rapid preparation of RNA from mammalian cell culture, laser capture micro-dissections and FACS-prepared cell populations, such as macrophages.

The kits 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 protects the sample and supports sample sizes down to single cells.

 

Kit components

 

RNAGEM™ Tissue PLUS RNAGEM™ Tissue
  •  RNAGEM enzyme   •  RNAGEM enzyme
  •  10 x SILVER buffer   •  10 x SILVER buffer
  •  RNAse-free DNAse 1   •  10 x TE storage buffer 
  •  10 x DNAse Buffer    •  QuickStart Guide
  •  10 x TE storage buffer   
  •  QuickStart Guide   

 

Preparation

All manipulations should be performed in a clean room or a PCR hood. Use only certified RNAse-free tubes and reagents and wash surfaces likely to come into contact with the samples in 0.05% hypochlorite bleach. Rinse thoroughly with RNAse-free water.

 

Range of Efficacy 

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.

 

A schematic of the extraction workflow is shown below.

RNAGEM Workflow

 

Extraction Method

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.