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Quantitation of Multiple RNA Species 211
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Quantitation of Multiple RNA Species
Ron Kerr
1. Introduction
Real-time quantitative polymerase chain reaction (PCR) methods (1) can be further
optimized for various purposes by performing quantitative PCR for multiple RNA
species in one sample (2). Advantages of this method not only include the elimination
of differences in reaction mix volumes and conditions that may occur in separate
samples, but importantly it also allows reference of RNA quantitation to an internal
control (also see Notes 1 and 2). Internal controls are RNA species for which the
quantity of RNA does not change across different cell types or conditions. They allow
correction of RNA quantitation for different starting quantities of total RNA. Widely
used examples of internal controls are 18S ribosomal RNA and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) (3), and commercial standard kits for these are
available. Different internal controls may be appropriate depending on the cell types
or conditions being studied, and it is advisable to try several different controls when
setting up a new assay. Quantitation of multiple RNA species is possible through the
use of fluorescent probes, such as Taqman probes, as previously described.
When multiple RNA species are to be quantified in a single sample, probes with
fluorophores of different wavelengths are used. As with any assay that relies on
the measurement of multiple fluorescent signals, it is important to ensure that the
wavelengths of the emitted signals are as distant as possible to reduce overlap in
signal detection (Fig. 1). The materials and methods for quantitation of mRNA for the
fibrinolytic protein tissue plasminogen activator (t-PA) using 18S ribosomal RNA as
an internal control are shown below as a worked example of the quantitation of two
RNA species in a single sample.
2. Materials
All reagents may be obtained from Applied Biosystems, Warrington, Cheshire, UK,
unless otherwise stated.
1. 100 ng/µL RNA sample (see Chapters 9–11 for RNA isolation method).
2. Reverse Transcriptase mastermix (9 µL per reaction): 2.85 µL of DEPC distilled H 2 O,
1 µL of 10× Taqman buffer, 2.2 µL of 25 mM MgCl 2 , 2 µL of dNTPs, 0.2 µL of RNase
Inhibitor, 0.25 µL of multiscribe RT, and 0.5 µL of random hexamers.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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