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Validating microarray data with real time rt pcr how to avoid dating an abusive creep

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In conventional real-time RT-PCR assays, expression is normalized to a control, or housekeeping gene.

However, no single housekeeping gene can be used for all studies.

RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been extensively analyzed.

Recently, the application of both microarrays and NGS has expanded to include micro RNAs (mi RNAs), but the relative performance of these methods has not been rigorously characterized.

validating microarray data with real time rt pcr-41validating microarray data with real time rt pcr-75validating microarray data with real time rt pcr-8

Differential expression is often validated using real-time reverse transcription PCR (RT-PCR) assays.

This challenge is partly addressed by ensuring that hybridization-based assays are performed at high enough temperatures to reject cross-hybridizing transcripts.

In addition, microarrays with probes containing locked nucleic acid (LNA) bases (Exiqon) provide higher annealing affinities, potentially allowing the assay to discern between individual mi RNA family members and somewhat equalizing the melting temperatures of probe sequences.

2008), all of which face unique challenges compared to their use in m RNA profiling.

In terms of microarray analysis, the short length of mature mi RNA sequences constrains probe design, such that often the entire mi RNA sequence must be used as a probe.