SAMPLE PREPARATION IN RNA ANALYSIS

Department of Pharmaceutical Chemistry, Rutgers University College of Pharmacy, 

Sample Preparation Techniques in Analytical Chemistry, Edited by Somenath Mitra ISBN 0-471-32845-6 Copyright 2003 John Wiley Sons, Inc. 

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This multiprobe RPA offers the advantages of its sensitivity and capacity to simultaneously quantitate several mRNA species in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples, moreover, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Furthermore, the assay is highly specific and quantitative owing to the RNase sensitivity of mismatched base pairs and the use of solution-phase hybridization driven toward completion by excess probe. Finally, the multiprobe RPA can be performed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells. 

Experimentally, temperature is used to systematically vary the RNA structure. When a short laser pulse is used to produce a rapid temperature increase in the sample, the structural changes that ensue can be followed in real time. In this contribution, we discuss experimental methods including sample preparation, instrumentation, and data analysis. We conclude with several experimental examples that highlight usefulness of the technique. 

The second section is dedicated to the preparation for nucleic acid analysis. Specific examples of DNA and RNA analyses are presented, along with the description of techniques used in these procedures. Sections on high-throughput workstations and microfabricated devices are included. The third section deals with sample preparation techniques used in microscopy, spectroscopy, and surface-enhanced Raman. 

CRITICAL ASSESSMENT OF THE METHOD Since differential expression analysis means to compare the quantities of RNA species in two samples, every step during sample preparation has to be highly reproducible. In order to maximize reproducibility, complete total RNA extraction in a one step procedure is recommended. Care must always be taken when working with RNA, to avoid contamination with RNA ases, which may result in RNA degradation. 

Figure 8 Automated high-throughput RNA analysis by capillary electrophoresis. Typical batch processing profiles of a 96-well sample plate. Total RNA sample preparations from rice (traces 1-76 from top), arabidopsis (traces 77-95), and yeast (trace 96) 6 pL each in 96-well plate. Conditions 50-pm-i.d. capillary, =10 cm (L = 30 cm) sieving medium, 1% PVP (polyvinylpirrolidone, MW= 1.3 MDa), 4 M urea, 1 xTBE, 0.5 pM ethidium bromide =500 V/cm 25°C. RNA samples were diluted in deionized water and denatured at 65°C for 5 min prior to analysis. Sample tray was stored at 4°C in the CE instrument during processing. Injection vacuum (5 s at 3.44 kPa). Separation matrix was replaced after each run, 2 min at 551 kPa. (Reproduced with permission from Ref. 102.)...

Recently it has been shown that the double-stranded RNA and its thermally denatured form behave in a similar manner to native and denaturated DNA [109]. Pulse-polarographic analysis in connection with a gel electrophoresis can be used for testing double-stranded RNA-samples prepared for biological experiments (interferon-inducing and antiviral activities). 

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