Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

Abstract

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<US$10) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a ß-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r 2 = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality. Key wordsAnalysis of the transcriptome using highdensity oligonucleotide arrays is an important tool for discovery in biology (1). The preparation of the target cRNA is a multistep procedure that requires RNA isolation, first strand cDNA synthesis with an oligo(dT) primer bearing a T7 promoter, second-strand cDNA synthesis, and a final in vitro transcription using T7 RNA polymerase and biotinylated ribonucleotides. These enzymatic reactions are sensitive to the quality of the starting RNA, the intermediate cDNA template, the occurrence of endogenous priming, and the presence of impurities, such as RNases. Incomplete cDNA or cRNA synthesis usually results in 5' shortening of the target cRNA molecules (2), which is difficult to assess using gel-based assays. To

Blood-Based Gene Expression Signatures in Non–Small Cell Lung Cancer

“…To determine the quality of cRNA, a semiquantitative reverse transcriptase PCR amplifying a 5 0 and a 3 0 product of the b-actin gene was used as previously described (14) and showed no sign of degradation with the 5 0 and a 3 0 product being present. All expression data presented in this article were of high quality.…”