Transcriptional expression profiling provided by the ncRNA Program is performed in one of two modes to derive the maximum amount of information in the most cost-effective manner. The Program offers two platforms from which to choose:
- Either SOLiD™ Next-Generation Sequencing (NGS), for whole transcriptome profiling, or Affymetrix GeneChip®, for limited genome-wide expression profiling, is first used when the fingerprint pattern and the identity of all possible expressed genes are sought.
- Targeted expression profiling is performed following genome-wide screening analysis and permits focus on a large number of replicated conditions, time points, cancer stages or samples from different individuals.
Analysis of tens of thousands of genes is a new challenge in molecular biology and requires the very careful design of experiments to obtain high quality results. Any technical variations will be observed in the results. When samples are compared for differential gene expression, care is taken to perform each step of the process in a similar fashion.
Examples of How Results can be Compromised
- Cultured cells should be seeded with the same density and culture medium in a Petri dish and treated in the same manner under the same conditions. Variable culture conditions will result in induction of the variable stress response, changes of cell cycle or apoptosis.
- Samples are treated differently before RNA isolation (storage, freezing). Heat shock or cold shock could be induced and apoptosis or RNA degradation could appear.
- Alterations in RNA isolation protocols (thawing of samples, usage of different methods of RNA isolation within a project, usage of mRNA and total RNA within a project). Artificially differentially expressed genes can be observed.
- Differences caused by variable RNA integrity due to technical variations (the ncRNA Program can quantify the RNA integrity of your samples). In an experimental comparison of pairs, stable mRNAs behave like upregulated differentially expressed genes and unstable RNAs behave like downregulated differentially expressed genes.
- Variations in chip processing (different batches of enzymes, several people doing the experiments). When minor alterations in gene expression are interrogated, these differences can completely camouflage biological differences.
Sample Quality and Quantity Requirements
Next Generation Sequencing
- DNA sequencing: 0.5~20 μg of DNA sample is needed for the application of whole genome re-sequencing, targeted genome sequencing, CHIP-sequencing and methylation profiling.
- RNA sequencing: 1~5 μg of intact total RNA is necessary for whole transcriptome sequencing or small RNA sequencing.
Affymetrix Genechip Applications
- mRNA expression profiling: 1~3 μg of intact total RNA, prepared by TRIzol® method, in RNase-free H2O at the concentration 0.5μg/μl as needed.
- SNP genotyping and CNV on Affymetrix Chips: 0.5~1.0 μg of genomic DNA as needed.
ncRNA/microRNA Expression Array
- microRNA expression profiling: 2~5 μg non-column purified total RNA extracted either from fresh tissue or FFPE tissues (0.5~1.0 μg/μl), prepared by TRIzol method or RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Ambion AM1975), in RNase-free H2O as needed.