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Influence of RNA labeling on expression profiling of microRNAs.

ABSTRACT: Although a number of technical parameters are now being examined to optimize microRNA profiling experiments, it is unknown whether reagent or component changes to the labeling step affect starting RNA requirements or microarray performance. Human brain/lung samples were each labeled in duplicate, at 1.0, 0.5, 0.2, and 0.1 ?g of total RNA, by means of two kits that use the same labeling procedure but differ in the reagent composition used to label microRNAs. Statistical measures of reliability and validity were used to evaluate microarray data. Cross-platform confirmation was accomplished using TaqMan microRNA assays. Synthetic microRNA spike-in experiments were also performed to establish the microarray signal dynamic range using the ligation-modified kit. Technical replicate correlations of signal intensity values were high using both kits, but improved with the ligation-modified assay. The drop in detection call sensitivity and miRNA gene list correlations, when using reduced amounts of standard-labeled RNA, was considerably improved with the ligation-modified kit. Microarray signal dynamic range was found to be linear across three orders of magnitude from 4.88 to 5000 attomoles. Thus, optimization of the microRNA labeling reagent can result in at least a 10-fold decrease in microarray total RNA requirements with little compromise to data quality. Clinical investigations bottlenecked by the amount of starting material may use a ligation mix modification strategy to reduce total RNA requirements.

SUBMITTER: Kaddis JS

PROVIDER: S-EPMC3338349 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Influence of RNA labeling on expression profiling of microRNAs.

Kaddis John S JS Wai Daniel H DH Bowers Jessica J Hartmann Nicole N Baeriswyl Lukas L Bajaj Sheetal S Anderson Michael J MJ Getts Robert C RC Triche Timothy J TJ

The Journal of molecular diagnostics : JMD 20111107 1

Although a number of technical parameters are now being examined to optimize microRNA profiling experiments, it is unknown whether reagent or component changes to the labeling step affect starting RNA requirements or microarray performance. Human brain/lung samples were each labeled in duplicate, at 1.0, 0.5, 0.2, and 0.1 μg of total RNA, by means of two kits that use the same labeling procedure but differ in the reagent composition used to label microRNAs. Statistical measures of reliability an ...[more]

PMID: 22074760

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Project description:MicroRNAs (miRNAs) are known to be deregulated in human breast cancer (BC). The purpose of the current study was to investigate the expression of miRNAs in different stages of BC to assess their biological value in BC progression. MiRNA expression was assessed in a series of BC patients (n=7) with distinct stages of tumour progression (Normal, in-situ (DCIS), primary invasive BC and nodal metastases) to evaluate miRNA differential expression. We used an Agilent miRNA microarray based platform which uses miRBase 16 to screen for 1205 Homo sapiens (hsa) and 144 human viral miRNA candidates. To validate the microarray data, the expression of two deregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). This study was conducted Formalin Fixed and Paraffin Embedded (FFPE) specimens from 7 female patients. Twenty μm thick FFPE sections were cut and mounted on PALM Membrane Slides. Under direct microscopic visualization, tissues of interest were micro-dissected using PALM non-contact Laser catapulsion instrument (P.A.L.M. Microlaser Technologies, Carl Zeiss Ltd). Total RNA, including miRNAs, was extracted with the TRIzol reagent (Invitrogen, supplied by Fisher Scientific UK Ltd) according to the manufacturer’s instructions. Total RNA was quantified with Quant-iT RiboGreen RNA Quantitation Kit (Fisher Scientific UK Ltd), which is an accurate method of measuring total RNA with a detection limit of 0.001-1ng/µl. RNA purity and RNA integrity number (RIN) were determined on an Agilent 2100 Bioanalyzer and RNA 6000 NanoLabChip Kits (both Agilent Technologies, Santa Clara, USA), and the RIN of all the samples was >2 but <3. This is expected for FFPE breast samples and is acceptable by Agilent Technologies, Santa Clara, USA, for FFPE samples to undergo miRNA microarray analysis. MiRNAs extracted from laser microdissected tissue components were profiled using Agilent microRNA microarray profiling system (Agilent Technologies, Santa Clara, USA). Total RNA samples were spiked using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) to evaluate efficiencies of labelling and hybridisation. Total RNA was treated with Calf Intestinal Alkaline Phosphatase (CIP), and then 100 ng of total RNA was used per sample to initiate a labelling reaction. Ligation master mix for T4 RNA ligase, which includes Cyanine 3-Cytidine biphosphate (Cyanine 3-pCp) (Complete miRNA Labelling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labelled miRNA samples were hybridised to human miRNA microarrays (Release 16.0, 8x60K) (Agilent Technologies, Santa Clara, USA) at 55°C for 20 hrs. Microarray slides were washed with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara), and subsequently dried with acetonitrile (Sigma-Aldrich, St. Louis, USA). Fluorescent signal intensities were detected on an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, USA) using the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from the images with the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).

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Influence of RNA labeling on expression profiling of microRNAs.

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Influence of RNA labeling on expression profiling of microRNAs.

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