Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation

Abstract

Background: Low RNA yields from clinical samples are a limiting step for microarray technology.

Aims: To design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the crucial step of global mRNA amplification performed before microarray hybridisation, using less than 1 microg total RNA.

Methods: Three RNA extraction procedures were compared for small size samples. Total RNA was amplified from universal RNA or the BC-H1 breast cancer micrometastatic cell line using three different protocols. Real time quantitative PCR technology was used for accurate measurement of urokinase plasminogen activator receptor and cytokeratin 8 RNA amplification rates and ratios, using primer sets binding at various distances from the 3' end of transcripts. A 50 mer oligomeric array targeting 87 genes potentially involved in breast cancer metastatic progression was built and hybridised with amplified RNA.

Results: Eighteen nanograms of total RNA could be purified from 1000 BC-H1 micrometastatic cells. Amplification rates of 25,000 to 100,000 were achieved with as little as 10 ng of starting material. However, results were highly variable, depending on the amount of starting material, gene characteristics, sample quality, and protocols used. Oligomeric array hybridisation with 20 microg reference RNA resulted in specific and reproducible signals for 83% of the genes, whereas mRNA amplification from less than 400 ng of starting material resulted in selective detection of signals from highly expressed genes.

Conclusions: Improvements in the design of global mRNA amplification procedures and oligomeric arrays are needed to extract informative gene expression data from clinical samples containing limited cell numbers.

Figure 1

Qualitative analysis of RNA before and after amplification. Total RNA extracted from (A) 10 000 BC-H1 micrometastatic cells, and amplified RNA ((B) one round or (C) two rounds) from 10 ng (purple), 100 ng (green), and 1000 ng (blue) BC-H1 total RNA, were tested for yield and integrity using the Agilent nanotechnology. Electropherograms and gel images show high quality mRNA amplification, with a decrease in amplified RNA size during the second round of amplification for the 100 ng sample.

Figure 2

Real time quantitative PCR strategy. (A) Name, sequence, and position in coding sequence (cds), for primer sets specific for the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cytokeratin 8 (Ker8), urokinase plasminogen activator receptor (uPAR), and the Arabidopsis thaliana rubisco activase (ATH rbcl) genes. (B) The above primer sets have been designed such that they bind at the 5′, the middle (mid), or the 3′ region of the coding sequence.

Figure 3

Real time quantitative polymerase chain reaction (rtqPCR) validation. (A) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rtqPCR amplification, (B) dissociation, and (C) standard curve plots, exemplify the methodology used to validate each primer set (human GAPDH O3-O4 here) and experimental conditions.

Figure 4

Real time quantitative polymerase chain reaction (rtqPCR) quantification of amplified RNA (aRNA) obtained with two T7 based protocols. Aliquots of 1 ng (yellow), 10 ng (green), and 100 ng (blue) universal RNA were amplified using (A, B) the Ambion and (C, D) the modified Affymetrix protocols. The first round (A, C) and the second round (B, D) of amplification were assessed, using rtqPCR with 3′ primers for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), urokinase plasminogen activator receptor (uPAR), and cytokeratin 8 (Ker8) genes. Amounts are expressed in nanograms and were calculated from standard curves for each primer set.

Figure 5

Assessment of the linearity of the global mRNA amplification procedure. Aliquots of 1 ng (yellow), 10 ng (green), and 100 ng (blue) universal RNA were subjected to (A) a first and (B) a second round of amplification, using the Ambion and modified Affymetrix (Affy-mod) protocols. Real time quantitative polymerase chain reaction was performed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 3′ primers, and amplification rates are reported as a ratio of the amount of amplified RNA produced with respect to the total RNA input.

Figure 6

Evaluation of 5′ truncation of the transcript during the T7 based amplification procedure. Aliquots of 1 ng, 10 ng, and 100 ng of uRNA were subjected to one round of amplification with the Ambion protocol. Real time quantitative polymerase chain reaction targeting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH; blue), urokinase plasminogen activator receptor (uPAR; purple), and cytokeratin 8 (Ker8; yellow) genes, with (A) 3′ and (B) 5′ primers, was used to measure the amplified RNA yield and to deduce amplification rates.