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RT PCR Protocols 1st Edition Nicola King Digital Instant
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Author(s): Nicola King, Joe OConnell
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Methods in Molecular Biology TM

VOLUME 193

RT-PCR
Protocols
Edited by
Joe O’Connell

HUMANA PRESS
RT-PCR Protocols
 METHODS IN MOLECULAR BIOLOGY TM

John M. Walker, SERIES EDITOR

220. Cancer Cytogenetics: Methods and Protocols, edited by John 190. High Throughput Screening: Methods and Protocols,
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201. Combinatorial Library Methods and Protocols, edited by 171. Proteoglycan Protocols, edited by Renato V. Iozzo, 2001
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193. RT-PCR Protocols, edited by Joe O’Connell, 2002 Acids, edited by Keith R. Mitchelson and Jing Cheng, 2001
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M E T H O D S I N M O L E C U L A R B I O L O G Y™

RT-PCR
Protocols

Edited by

Joe O’Connell
Department of Medicine,
National University of Ireland,
Cork, Ireland

Humana Press Totowa, New Jersey

©2002 Humana Press Inc.
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Production Editor: Kim Hoather-Potter.

Cover design by Patricia F. Cleary.

Cover illustration: Background from Fig. 3A in Chapter 17 “RT-PCR-Based Approaches to Generate Probes
for mRNA Detection by In Situ Hybridization” by Joe O’Connell; foreground from Fig. 2 in Chapter 18
“Amplified RNA for Gene Array Hybridizations” by Valentina I. Shustova and Stephen J. Meltzer.

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Library of Congress Cataloging in Publication Data

RT-PCR Protocols/edited by Joseph O’Connell

p.cm.-- (Methods in molecular biology)
Includes bibliographical references and index.
ISBN 0-89603-875-0 (alk. paper)
1. Polymerase chain reaction--Laboratory manuals. I. O’Connell, Joseph. II. Methods in
molecular biology (Clifton, N.J.); v. 193.

QP606.D46 R8 2002
572'.43--dc21
2002190221
Preface
Until the mid 1980s, the detection and quantification of a specific mRNA
was a difficult task, usually only undertaken by a skilled molecular biologist.
With the advent of PCR, it became possible to amplify specific mRNA, after
first converting the mRNA to cDNA via reverse transcriptase. The arrival of
this technique—termed reverse transcription-PCR (RT-PCR)—meant that
mRNA suddenly became amenable to rapid and sensitive analysis, without
the need for advanced training in molecular biology. This new accessibility of
mRNA, which has been facilitated by the rapid accumulation of sequence data
for human mRNAs, means that every biomedical researcher can now include
measurement of specific mRNA expression as a routine component of his/her
research plans.
In view of the ubiquity of the use of standard RT-PCR, the main objective
of RT-PCR Protocols is essentially to provide novel, useful applications of
RT-PCR. These include some useful adaptations and applications that could
be relevant to the wider research community who are already familiar with the
basic RT-PCR protocol. For example, a variety of different adaptations are
described that have been employed to obtain quantitative data from RT-PCR.
Quantitative RT-PCR provides the ability to accurately measure changes/imbal-
ances in specific mRNA expression between normal and diseased tissues.
Because of its remarkable sensitivity, RT-PCR enables the detection of low-abun-
dance mRNAs even at the level of individual cells. RT-PCR has afforded many
opportunities in diagnostics, allowing sensitive detection of RNA viruses such as
HIV and HCV. RT-PCR facilitates many diverse techniques in research, includ-
ing in situ localization of mRNA, antibody engineering, and cDNA cloning. In
particular, the present work highlights how RT-PCR complements other tech-
nological advances, such as laser-capture microdissection (LCM), real-time
PCR, microarray technology, HPLC, and time-resolved fluorimetry.
RT-PCR has become one of the most widely applied techniques in bio-
medical research, and has been a major boon to the molecular investigation of
disease pathogenesis. Determination of the pathogenesis of diseases at the
molecular level is already beginning to inform the design of new therapeutic
strategies. It is our hope that RT-PCR Protocols will stimulate the reader to
explore diverse new ways in which this remarkable technique can facilitate
the molecular aspects of their biomedical research.
Joe O’Connell

v
Contents
Preface ............................................................................................................. v
Contributors ..................................................................................................... xi

PART I. INTRODUCTION
1 RT-PCR in Biomedicine: Opportunities Arising from the New
Accessibility of mRNA
Joe O’Connell ......................................................................................... 3
2 The Basics of RT-PCR: Some Practical Considerations
Joe O’Connell ....................................................................................... 19
PART II. HIGHLY SENSITIVE DETECTION AND ANALYSIS OF MRNA

3 Using the Quantitative Competitive RT-PCR Technique

to Analyze Minute Amounts of Different mRNAs in Small
Tissue Samples
Susanne Greber-Platzer, Brigitte Balcz,
Christine Fleischmann, and Gert Lubec ..................................... 29
4 Detection of mRNA Expression and Alternative Splicing
in a Single Cell
Tsutomu Kumazaki .............................................................................. 59
5 Nested RT-PCR: Sensitivity Controls are Essential
to Determine the Biological Significance of Detected mRNA
Triona Goode, Wen-Zhe Ho, Terry O’Connor,
Sandra Busteed, Steven D. Douglas, Fergus Shanahan,
and Joe O’Connell .......................................................................... 65
PART III. QUANTITATIVE RT-PCR
6 Quantitative RT-PCR: A Review of Current Methodologies
Caroline Joyce ..................................................................................... 83
7 Rapid Development of a Quantitative-Competitive (qc)
RT-PCR Assay Using a Composite Primer Approach
Joe O’Connell, Aileen Houston, Raymond Kelly,
Darren O’Brien, Aideen Ryan, Michael W. Bennett,
and Kenneth Nally .......................................................................... 93
8 Quantitation of Gene Expression by RT-PCR and HPLC
Analysis of PCR Products
Franz Bachmair, Christian G. Huber,
and Guenter Daxenbichler ............................................................ 103

vii
viii Contents

9 Time-Resolved Fluorometric Detection of Cytokine mRNAs

Amplified by RT-PCR
Kaisa Nieminen, Markus Halminen, Matti Waris, Mika Mäkelä,
Johannes Savolainen, Minna Sjöroos, and Jorma Ilonen ...... 117
10 Mimic-Based RT-PCR Quantitation of Substance P mRNA
in Human Mononuclear Phagocytes and Lymphocytes
Jian-Ping Lai, Steven D. Douglas, and Wen-Zhe Ho ..................... 129
PART IV. DETECTION AND ANALYSIS OF RNA VIRUSES
11 Detection and Quantification of the Hepatitis C Viral Genome
Liam J. Fanning ................................................................................. 151
12 Semi-Quantitative Detection of Hepatitis C Virus RNA
by "Real-Time" RT-PCR
Joerg F. Schlaak ................................................................................ 161
13 RT-PCR for the Assessment of Genetically Heterogenous
Populations of the Hepatitis C Virus
Brian Mullan, Liam J. Fanning, Fergus Shanahan,
and Daniel G. Sullivan ................................................................. 171
PART V. IN SITU LOCALIZATION OF MRNA EXPRESSION
14 In Situ Immuno-PCR: A Newly Developed Method for Highly
Sensitive Antigen Detection In Situ
Yi Cao .................................................................................................. 191
15 RT-PCR from Laser-Capture Microdissected Samples
Tatjana Crnogorac-Jurcevic, Torsten O. Nielsen,
and Nick R. Lemoine .................................................................... 197
16 Mycobacterium paratuberculosis Detected by Nested PCR
in Intestinal Granulomas Isolated by LCM in Cases
of Crohn’s Disease
Paul Ryan, Simon Aarons, Michael W. Bennett, Gary Lee,
Gerald C. O’Sullivan, Joe O’Connell,
and Fergus Shanahan .................................................................. 205
17 RT-PCR-Based Approaches to Generate Probes for mRNA
Detection by In Situ Hybridization
Joe O’Connell ..................................................................................... 213
PART VI. DIFFERENTIAL MRNA EXPRESSION
18 Amplified RNA for Gene Array Hybridizations
Valentina I. Shustova and Stephen J. Meltzer .............................. 227
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 should come. Or, it may, perhaps, mean that they were
individually to treasure it up, having designated in their own
minds the sum which they could give, and have it in readiness
when he should come.”

8. Instead of selecting a wealthy person, able to contribute ten

dollars per week, as has been done above, let an individual be
chosen from the poorer classes of Corinthians—say from
among these who would be able to donate only twenty-five
cents per week—and the reader will be more forcibly
impressed with the unreasonableness of that construction
which makes it necessary that so small a pittance should first
be placed or devoted at home, and then carried to the church,
and there deposited in the general collection.

9. Not a few eminent writers, such as Dwight, and Wilson, of

Calcutta, who are followed by many lesser authors, quote
Ignatius, as saying: “Let us no more Sabbatize, but keep the
Lord’s day.” From the literal rendering of the original above
given, it will be seen that these writers take an unwarrantable
liberty with their author. The words of Ignatius are, ἀλλὰ κατὰ
τὴν κυριακὴν ζωὴν ζῶντες. To separate the noun ζωὴν from
the preceding adjective, and connect it with the following
participle, so as to read, “Living a life according to the Lord’s
day,” is an unnatural separation of the words of the original. To
drop out the word ζωὴν is unwarranted. If this word were
spurious, then the rendering would be, “Living according to the
Lord’s day,” the adjective κυριακη without the noun for “day”
being expressed occurring frequently for “the Lord’s day.” But
there is no ground for rejecting the word “life.” To color the
language of an author for the sake of giving it point in favor of
one side of a question is unworthy of a seeker after truth. In
the present case there is really nothing gained by departing
from the precise language of the writer. Another passage,
often quoted as from Ignatius, is part of the spurious epistle to
the Galatians. It is as follows: “During the Sabbath, Christ
 continued under the earth, in the tomb in which Joseph of
Arimathea had laid him. At the dawning of the lord’s day, he
arose from the dead. The day of the preparation, then,
comprises the passion; the Lord’s day contains the
resurrection.” This certainly has some weight as the testimony
of comparatively early writer, but it must not be ascribed to
Ignatius.

10. Did it not appear to be indispensable to the enlightening of the

reader, as to the consummate folly of the author of the epistle
of Barnabas, we should not append, as we do, his language in
the following note, since it is hardly worthy of a place in a
chaste and dignified discussion. For its citation we hold those,
responsible who have made this action necessary, and who
value the testimony of a man so utterly devoid of common-
sense: “Neither shalt thou eat of the hyena; that is, again, be
not an adulterer; nor a corrupter of others; neither be like to
such. And wherefore so? Because that creature every year
changes its kind, and is sometimes male and sometimes
female.” Chap. 9:8.

11. Since, writing the above, the following interesting item in the
Christian Union, for Feb. 19, has been brought to my notice,
and will serve to show that continued investigation on the part
of scholars is rendering the authenticity of the writings of
Justin Martyr more and more doubtful:—“Dr. Franz Overbeck
has lately examined, with great care, the ‘epistle to Diognetus,’
which has been regarded as one of the most precious relics of
the age succeeding that of the apostles. He urges several
reasons for coming to the conclusion that the work was written
later than the era of Constantine, and was intended by its
author to pass as a work Justin Martyr’s. Critics had already
proved it no genuine work of Justin, and if Dr. Overbeck is
right, it can no longer be assigned to the age of Justin.”
12. The culpable carelessness of Dwight, Wilson, and other
authors, in citing from the early fathers, is nowhere more
noticeable than in the case of Irenæus. These writers quote
him as saying: “On the Lord’s day, every one of us Christians,
keeps the Sabbath, meditating on the law, and rejoicing in the
works of God.” There is no reference given to the writings of
Irenæus. And for good reason. After a most careful
examination, we are persuaded no such passage is to be found
in his writings. The mistake was probably first made by
President Dwight, whose weakness of sight compelled him to
depend upon an amanuensis. “For twenty years of his
presidency,” we are informed by his biographer, “he was rarely
able to read as much as a single chapter in the Bible in the
twenty-four hours.” (Dwight’s Theology, London, 1821, vol. i.
pp. 91, 95.) Others followed this high authority.
In order to guard our readers against injuring the cause they
would advance, we must mention another important instance
of considerable negligence. In a number of works on the
Sabbath, Dr. Justin Edwards’ “Sabbath Manual,” for example,
we find not only the blunders already noticed, but another
quite as bad. The language—“Both custom and reason
challenge from us that we should honor the Lord’s day, seeing
on that day it was that our Lord Jesus completed his
resurrection from the dead,” is ascribed to Theophilus, bishop
of Antioch, about A. D. 162. The words quoted are in reality
those of another Theophilus, who was bishop of Alexandria, at
the close of the fourth century. We hand over these criticisms
upon advocates of the first-day Sabbath to our seventh-day
Sabbatarian friends, trusting to their honor and fairness not to
separate them from the rest of this discussion. For our own
part, whether it may be pleasant to the advocates of the
seventh-day Sabbath, we desire to have for ourselves, and to
aid others to have, the whole truth. It was in this spirit that we
gave room in our columns for a full presentation of the
arguments on the other side of this question.
13. As a matter of independent interest and importance, we would
ask all who are interested in the question of the posture in
prayer of worshipers in the early church, to compare with
Tertullian’s statement, that of Peter, bishop of Alexandria, A. D.
300, who says: “We keep the Lord’s day as a day of joy,
because of Him who rose on that day, on which we have
learned not to bow the knee.” (Bibl. Patrum, apud Gallard, vol.
iv., p. 107.) To the same effect is the decision of the Council of
Nice, A. D. 325, requiring, as there were certain ones who bent
the knee on the Lord’s day, that it should be the uniform
practice to give thanks to God, standing. (Canon, xx.)

14. The attempt to attribute the change of day to Constantine’s

decree is hardly worth noticing. It is enough to remember that
it was issued in the beginning of the fourth century. No one
who knows anything of the writings of Tertullian and Origen
dating back more than a century before Constantine, to say
nothing of still earlier writers, will venture to ascribe the
change to Roman Emperor’s decree. Besides, the language of
the very decree referred to recognizes the honorable diameter
of the first day of the week. It recognizes that day as already
“venerable.”—The Christian.

15. For the extracts given in this connection, the reader is referred
to “Sabbath and Sunday,” by A. H. Lewis, and to “The History
of the Sabbath,” by J. N. Andrews.

16. The commandments as given above are supposed to be

repeated by the individual Romanist in response to the
injunction, “Say the ten commandments of God.”

17. The following citations will be found in a small tract published

at the “Review and Herald” Office, entitled, “Who Changed the
Sabbath?”
18. By consulting the figures given above, the reader will be able
to demonstrate, not only the fact that the inhabitants along the
line from Pekin to San Francisco, can hallow the same day, but
also that the day which they hallow will be identical in some of
its hours. For example: It was shown that the people of Rome
commence their day six hours and fifty-five minutes later than
do those of Pekin. Deducting these six hours and fifty-five
minutes from twenty-four hours we have left seventeen hours
and five minutes as the period of time during which the
citizens of these two cities would be celebrating the Sabbath in
common. Applying the same principle to other cities, we find
that London and Pekin would worship together for sixteen
hours and fifteen minutes; New York and Pekin, eleven hours
and twenty minutes; Chicago and Pekin, ten hours and twenty-
five minutes; S. Francisco and Pekin, eight hours and five
minutes.

19. The gentleman might have cited the case of Alaska, also, as a
parallel to that of Pitcairn’s Island. The inhabitants of this
region, like those of the island mentioned, sailed eastward to
this continent across the Pacific Ocean, and failed to drop the
required day in their reckoning. The result was, that when we
purchased that territory, they were found to be keeping
Saturday instead of Sunday. We believe, however, that the
mistake is now rectified.
Transcriber’s Notes:
Missing or obscured punctuation was silently corrected.
Typographical errors were silently corrected.
Inconsistent spelling and hyphenation were made consistent
only when a predominant form was found in this book.
Footnotes have been collected at the end of the text, and are
linked for ease of reference.
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