|Position:||Post. doc., PhD|
|Phone:||+47 2278 1781|
1. Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance.
2.Gene expression analysis of a SMZL lymphoma patient panel. ApplyingmRNA amplification as an approach to obtain gene expression data frompatient samples with limited material.
3. Effects of furtherreduction of input RNA into the mRNA amplification protocol. Analysisof data set obtained from 10 000 cells vs 1000 cells, and 10 000 vs 250cells. Questions: Are the relative transcript levels maintained assamples are reduced? Can we obtain reliable data from as few as 250cells using the present amplification protocol.
Microarraytechnology provides high-throughput analysis of gene expressionprofiles. However, using standard protocols, a substantial amount ofRNA (10-50mg) is needed in order to apply this powerful technology.This molecular approach is thus rendered useless for many purposes ifthis limitation is not overcome.
The increasing need tobroaden the use of microarray technology has resulted in approaches toamplify the target RNA. Global amplification of RNA has been developedand used in gene expression analysis before the microarray techniqueappeared (Eberwine et al ., 1990). In this method, mRNA is reversetranscribed with an oligo dT/T7 promoter primer. After synthesis ofdouble stranded cDNA, antisense RNA is transcribed in vitro generallyresulting in a 1000-fold amplification of the original amount. Ifnecessary, a second round of amplification can be performed simply byrepeating the procedure. The second round is primed with randomhexameres, resulting in aRNA with reduced transcript lengths. Dataresults are not affected as long as probes on the microarrays also are3’ biased.
Atthe present this approach appears to be the most promising in regardsto gene expression analysis on material derived from sensitiveselection techniques such as immunomagnetic bead selection (IMS) andlaser capture micro dissection (LCM).
We have adopted 2 roundsof amplification as a standard and follow the protocol described byBaugh et al., (2002) with some minor enzyme modifications.
GlobalmRNA amplification has been applied in several publications, howeverthere has been minimal quantitative documentation of the linearity ofthe amplification procedure. We performed statistical analysis(multiple hypothesis testing and ANOVA variance analysis) on a data setgenerated from amplified and non-amplified material. We found a valueof less than 10% false discovery rate of significantly differentiallyexpressed genes when comparing the two data sets (amplified vs.non-amplified). From the ANOVA, we concluded that the variance due toamplification was relatively low compared to the overall variance. Forfurther reading see Nygaard et al., (2003) BMC Genomics.
Nygaard web page
Methods for quantitation of gene expression
Front Biosci, 14, 552-69
Cell sampling and global nucleic acid amplification
SEB Exp Biol Ser, 61, 17-36
Validation of oligoarrays for quantitative exploration of the transcriptome
BMC Genomics, 9, 258
Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling
Nucleic Acids Res, 34 (3), 996-1014
Limitations of mRNA amplification from small-size cell samples
BMC Genomics, 6, 147
Constitutive expression of the AP-1 transcription factors c-jun, junD, junB, and c-fos and the marginal zone B-cell transcription factor Notch2 in splenic marginal zone lymphoma
J Mol Diagn, 6 (4), 297-307
ABI3 mediates expression of the peroxiredoxin antioxidant AtPER1 gene and induction by oxidative stress
Plant Mol Biol, 53 (3), 313-26
MArray: analysing single, replicated or reversed microarray experiments
Bioinformatics, 18 (8), 1139-40