Moderate degradation does not preclude microarray analysis of small amounts of RNA

Abstract

Gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques. The quality of RNA preparations from such samples constitutes a frequent issue in this context. The aim of this study was to assess the impact of different extents of RNA degradation on the expression profile of the samples. We induced RNA degradation in human tumor and healthy tissue samples by endogeneous ribonucleases. Next, we amplified 20 ng total RNA degraded to different extents by two rounds of in vitro transcription and analyzed them using Affymetrix oligonucleotide microarrays. Expression differences for some genes were independently confirmed by real-time quantitative PCR. Our results suggest that gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity compared to intact samples and that RNA samples of suboptimal quality might therefore still lead to meaningful results if used carefully.

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

“…Fragmentation of long mRNA will result in a loss of the molecule for qPCR detection only if the RNA break occurs within the product sequence. This might be a rare event in only moderately degraded RNA (Schoor et al 2003). Therefore, the sequence of b-actin was assessed in different tissues and by varying RNA integrity.…”

“…Long mRNA, up to several kilo bases, is very sensitive to degradation (Bustin 2002). This can occur through cleavage by RNAses during handling of RNA samples, otherwise it may also be impaired in samples stored for a long time or under sub-optimal conditions (Schoor et al 2003). Consequently, the determination and confirmation of RNA quantity and quality is the first critical step in obtaining meaningful gene expression data.…”

Validation of adequate endogenous reference genes for reverse transcription-qPCR studies in human post-mortem brain tissue of SIDS cases

“…RNA degradation can occur due to various reasons such as natural fragmentation, inadequate sample handling, suboptimal storage conditions and others, especially when dealing with post-mortem samples [40]. The most common procedure nowadays to assess RNA integrity is the estimation of the RIN (RNA Integrity Number).…”

“…Spectrophotometer readings estimated that RNA quality and purity of all samples were acceptable and more importantly they did not differ between the SIDS and the control groups. In the reverse transcription step, as expression differences obtained from degraded samples using random-primed cDNA can correlate with high quality samples [40], a mix of random hexamer primers with oligo (dT) primers was used to insure priming at random sites throughout the RNA strand in addition to the poly (A) tail priming and thus decrease the effect of any possible RNA degradation on cDNA synthesis by capturing all RNA fragments.…”

“…Schoor et al [40] and van der Linden et al [47] stated that while all efforts should be made to obtain high quality RNA samples that reflect the natural state most reliably, partially degraded samples may still lead to a reasonable expression profile. Therefore, due to all of these aspects, we believe that RNA integrity did not have a high impact in the expression profiling and analysis of the present work.…”