Expression Profiling using low Quantity Samples

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, the GPCF has implemented two protocols based on a non-exponential amplification method using T7 in vitro transcription (IVT) technology. It allows representative amplification of as little as 500 picogrames of total RNA, followed by chemical labelling of the amplification product. Samples prepared this way can be combined with any human or mouse Affymetrix array.

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

  • Completion of sample sheet
  • Amount of RNA 3-100 ng
  • Concentration of >0.5 ng/µl
  • Volume > 6µl
  • 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

All samples should be treated with DNase.

Samples must be non-pathogenic and non-infectious (S1-Condition).

RNA samples must be provided in a 1.5ml-tube and on dry-ice

Please consult the pricelist for detailed cost information. 

 

We perform

  • Assistance with Experimental Design
  • QC of input RNA (Bioanalyzer Picochip)
  • Labeling and hybridization to the Affymetrix microarrays you request
  • Monitoring the quality at all steps
  • Basic Data Analysis

 

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
  • Group Comparisons

  

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.

 

Citing our service

Please cite our service in the acknowledgements part of your paper in case we've just performed the standard service.

last update:
24/04/2019
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