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Related Experiment Videos

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA.

Ruchira Singh1, Rajanikanth J Maganti, Sairam V Jabba

  • 1Dept. of Anatomy & Physiology, College of Veterinary Medicine, Kansas State Univ., 1600 Denison Ave., Coles Hall 205, Manhattan, KS 66506, USA.

American Journal of Physiology. Cell Physiology
|December 23, 2004
PubMed
Summary

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The picogram RNA sample (pRS) system effectively amplifies gene expression from minimal RNA amounts, detecting most genes and offering a faster, more stable alternative to T7-based methods for microarray analysis.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Microarray gene expression profiling typically requires microgram RNA quantities.
  • This requirement limits its use with nanogram RNA samples from techniques like microdissection or laser capture microscopy.

Purpose of the Study:

  • To evaluate novel RNA amplification and labeling systems for low-input microarray analysis.
  • To compare the performance of Ribo-SPIA (RS), an optimized prototype (pRS), and two T7-based amplification methods (One RA, Two RA).

Main Methods:

  • Analysis of mouse kidney and universal reference RNA samples (0.3 ng to 10 µg) using Affymetrix Mouse Genome 430 2.0 GeneChip arrays.
  • Evaluation of call concordance, signal intensity correlations, and fold-change detection across different amplification systems.

Related Experiment Videos

  • Comparison of amplification efficiency, gene detection rates, and technical simplicity.

    • Main Results:

    • All evaluated systems amplified overlapping gene sets with similar signal intensity correlations.
    • The pRS system amplified the highest number of genes from 10-ng RNA samples and detected 24 of 26 RT-PCR verified genes.
    • Two RA showed slightly higher call concordance (91.8%) compared to RS (89.3%) and pRS (88.1%).
    • pRS is suitable for amplifying as little as 0.3 ng of total RNA.

    Conclusions:

    • The pRS system is highly effective for amplifying nanogram RNA samples for gene expression profiling.
    • RS and pRS offer faster, simpler workflows and produce more stable cDNA compared to T7-based methods.
    • These optimized systems significantly expand the utility of microarrays for studies using limited RNA quantities.