Main description:

Once a tedious, highly skilled operation, reverse-transcription polymerase chain reaction (RT-PCR) has become a routine and invaluable technique used in most laboratories. In RT-PCR Protocols, Second Edition, expert researchers fully update the technologies presented in the popular previous edition, such as competitive RT-PCR, nested RT-PCR, RT-PCR from single cells, and RT-PCR for cloning. In addition, newer technologies are also explored, including multiplex RT-PCR, RT-LATE-PCR, and the greatly advanced field of real-time quantitative RT-PCR, while recent advances in creating the optimum RT-PCR reaction, e.g. RNA extraction, primer design, and reverse transcription, end the book with their indispensable input. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes sections, highlighting tips on troubleshooting and avoiding known pitfalls.

User friendly and up-to-date, RT-PCR Protocols, Second Edition acts as a handy companion to scientists from numerous diverse backgrounds who wish to explore further the marvels of gene expression.

Contents:

Part I: The RT-PCR Detective: Hunting Down the Best Method

1. Single Cell RT-PCR on Mouse Embryos: A General Approach for Developmental Biology
Wolfgang Mann and Thomas Haaf

2. Poly(A) cDNA Real-Time PCR for Indicator Gene Measurement in Cancer
Melissa Oliveira-Cunha, Ajith K. Siriwardena, and Richard J. Byers

3. Transcriptome Profiling of Host-Microbe Interactions by Differential Display RT-PCR
W. F. Leong and Vincent T. K. Chow

4. Quantitative RT-PCR Methods for Mature microRNA Expression Analysis
Stephanie D. Fiedler, Martha Z. Carletti, and Lane K. Christenson

5. Detection of Influenza A Virus Neuraminidase and PB2 Gene Segments by One Step Reverse Transcription Polymerase Chain Reaction
Alejandra Castillo Alvarez, Victoria Boyd, Richard Lai, Sandy Pineda, Hans G. Heine, and Ross Barnard

6. Detection and Identification of CD46 Splicing Isoforms by Nested RT-PCR
Anita Szalmas, Jozsef Konya, Istvan Sziklai, and Tamas Karosi

7. Simultaneous Detection of Bluetongue Virus RNA, Internal Control GAPDH mRNA, and External Control Synthetic RNA by Multiplex Real-Time PCR
Frank Vandenbussche, Elise Vandemeulebroucke, and Kris De Clercq

8. Detection of West Nile Viral RNA from Field-Collected Mosquitoes in Tropical Regions by Conventional and Real-Time RT-PCR
Ana Silvia Gonzalez-Reiche, Maria de Lourdes Monzon-Pineda, Barbara W. Johnson, and Maria Eugenia Morales-Betoulle

9. Detection of Antisense RNA Transcripts by Strand-Specific RT-PCR
Eric C.H. Ho, Michael E. Donaldson, and Barry J. Saville

10. RT-PCR Amplification and Cloning of Large Viral Sequences
Xiaofeng Fan and Adrian M. Di Bisceglie

Part II: The RT-PCR Mathematician: Assessing Gene and RNA Expression

11. One-Step RT-LATE-PCR for mRNA and Viral RNADetection and Quantification
Cristina Hartshorn and Lawrence J. Wangh

12. Changes in Gene Expression of Caveolin-1 after Inflammatory Pain Using Quantitative Real-Time PCR
Fiza Rashid-Doubell

13. Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction
Hongxin Fan and Ryan S. Robetorye

14. The Use of Comparative Quantitative RT-PCR to Investigate the Effect of Cysteine Incubation on GPx1 Expression in Freshly Isolated Cardiomyocytes
Nicola King

15. The Renaissance of Competitive PCR as an Accurate Tool for Precise Nucleic Acid Quantification
Lorena Zentilin and Mauro Giacca

Part III: The RT-PCR Master Chef: Finding the Right Ingredients

16. Skeletal Muscle RNA Extraction in Preparation for RT-PCR
Janelle P. Mollica

17. Reverse Transcription of the Ribonucleic Acid: A First Step in RT-PCR Assay
Fadia Haddad and Kenneth M. Baldwin

18. Primer Design for RT-PCR
Kelvin Li and Anushka Brownley

19. Hot Start PCR
Natasha Paul, Jonathan Shum, and Tony Le

20. Real-time RT-PCR for Automated Detection of HIV-1 RNA During Blood Donor Screening
Jens Muller