The need for a significant amount of starting RNA in expression profiling has limited its use in applications where the amount of RNA is low, such as with Laser Captured Microdissection (LCM), small biopsies, sorted cells or stem cell biology. To overcome this limitation, various RNA amplification and labeling methods have been described that start with low nangram amounts of total RNA. Techniques include T7 polymerase amplification methods (van Gelder et al., 1990, Wang et al., 2000, Baugh et al., 2001), PCR-based methods (Hertzberg et al., 2001, Iscove et al., 2002, Li et al., 2003) and single primer amplification technologies (Smith et al., 2003). However, most of these technologies either lack robustness/reproducibility or require cumbersome and lengthy handling procedures that cannot be standardized to a level acceptable for routine service. Recently the GPCF has adopted a non-exponential amplification method developed by nuGEN (Dafforn et al., 2004). It allows representative amplification of as little as 3 nanogrames of total RNA, followed by chemical labelling of the amplification product. The method is based on the Ribo-SPIA Technology (see figure).
In the first step first strand cDNA is generated that contains a unique RNA sequence at the 5’ end of the cDNA strand. In the second step a DNA/RNA heteroduplex double strand cDNA is generated which results in a double-stranded cDNA with a unique DNA/RNA heteroduplex at one end. In the third step the SPIA amplification is performed. In this step RNaseH is used to degrade RNA in the DNA/RNA heteroduplex at the 5’end of the first cDNA strand in order to expose the DNA for binding a second SPIA DNA/RNA chimeric primer. This step is repeated several times and results in a linear amplification of the target (figure courtesy of nuGEN Technologies Inc.)
The technology has matured to a level that samples prepared this way can be combined with any human, mouse or rat Illumina array, allowing for genome-wide expression profiling with as few as 3 nanograms.
Please take into consideration that the technical reproducibility is reduced when working with low quantity samples, and make use of additional biological replicates, compared to classical expression profiling. Moreover, it is not possible to compare results of classical expression profiling with the low quantity protocol. Please plan your projects accordingly.
GPCF will apply the low quantity protocol for all samples that are submitted at less than 100 ng total RNA.
YOU supply
- Amount of RNA 3-100 ng
- Concentration of >0.5 ng/µl
- RNA index >7
- Low quantity protocol will automatically be used when less than 100 ng of otal RNA are submitted for profiling - nos specific selection in the submission form is necessary
- We recommend to run at least twice as many biological replicates as compared to classical expression profiling
What we do
- QC of input RNA (Bioanalyzer Picochip)
- Synthesis of double stranded cDNA
- SPIA amplification of cDNA
- Chemical labeling of amplified material
- Hybridization to specified Sentrix Beadarray
- Image analysis
- Overall QC
Output to YOU
- All RNA and labeling QC data (conc. of input RNA, quality of input RNA, conc. of double stranded cDNA, conc. of labeled cDNA)
- Raw data of microarrays
- Individual chip QC (for chip integrity and hybridization success)
- Raw data evaluation (box plots)
- Interpreted data (numerical readout and gene annotation for microarray elements (probe sets)
- Normalization across each chip
- Normalization (if wanted) for complete sets of experiments
What's next?
You may unlock the insights buried in your experimental data, applying statistical analysis using DKFZ' in-house Chipster installation that makes use of R and Bioconductor packages and a Java GUI. Pathway analysis tools like Ingenuity Pathway Analysis or DAVID are also available via direct links, or provision of a computer workstation at GPCF (booking system for Ingenuity). Check out our webpage for more details and to get access.
For more Information, please review the FAQ site on expression profiling and the respective glossary.
Please consult the pricelist for detailed cost information.
