Skip to main content

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR


We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead.


  1. Webb T. Laser Capture Microdissection Comes Into Mainstream Use. J Natl Cancer Inst 2000; 92(21):1710–1711.

    Article  PubMed  CAS  Google Scholar 

  2. Invitrogen Life Technologies Instruction Manual. SuperScript III CellsDirect cDNA Synthesis System (for Catalog Nos. 18080-200 and 18080-300) Version A, 25-0731 14 May 2004; Version A; 1–5.

  3. Use of glycogen or tRNA for reducing non-specific adherence of templates to Plastic. Applied Biosystems Incorporated 5700 User Manual 2000. Section 5, p.14.

  4. Lee J, Duncan A, Warrick S. Techniques for Optimizing RT-PCR Reactions. PharmaGenomics 2003: pp. 54–55.

  5. Dunphy J, Horvath A, Barcham G, Balic A, Bischof R, Meeusen E. Isolation, characterisation and expression of mRNAs encoding the ovine CC chemokines, monocyte chemoattractant protein (MCP)-1α and -2. Vet Immunol Immunopathol 2001; 82(3-4):153–164.

    Article  PubMed  CAS  Google Scholar 

  6. Grubor B, Gallup JM, Meyerholz DK, Crouch EC, Evans RB, Brogden KA, Lehmkuhl HD, Ackermann MR. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs. Clin Diagn Lab Immunol 2004; 11(3):599–607.

    Article  PubMed  CAS  Google Scholar 

  7. Applied Biosystems User Bulletin #2, ABI PRISM 7700 Sequence Detection System. SUBJECT: Relative Quantitation of Gene Expression: “Calculating the Input Amount.” 2001. p. 8.

  8. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29(9):e45.

    Google Scholar 

  9. Bustin SA. A-Z of Quantitative PCR. Bustin SA, editor. (IUL Biotechnology, No. 5), 1st ed. International University Line; August 2004. 882 pages.

  10. Mocellin S, Rossi CR, Pilati P, Nitti D, Marincola FM. Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 2003; 9(5):189–195.

    Article  PubMed  CAS  Google Scholar 

  11. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 2000; 18(4):457–459.

    Article  PubMed  CAS  Google Scholar 

  12. Gallup JM, Grubor B, Barua AB, Mohammadi G, Brogden KA, Olson JA, Mark R, Ackermann MR. Repeated intravenous doses of all-trans-retinoyl β-Dglucuronide is not effective in the treatment of bacterial bronchopneumonia in lambs but is devoid of gross and acute toxicity. Med Sci Monit 2002; 8(9):BR345-BR353.

    PubMed  CAS  Google Scholar 

  13. Caverly JM, Diamond G, Gallup JM, Brogden KA, Dixon RAF, Ackermann MR. Coordinated Expression of Tracheal Antimicrobial Peptide and Inflammatory Response Elements in the Lungs of Neonatal Calves and with Acute Bacterial Pneumonia. Inf Immun 2003; 71(5):2950–2955.

    Article  CAS  Google Scholar 

  14. Opriessnig T, Yu S, Gallup JM, Evans RB, Fenaux M, Pallares FJ, Thacker EL, Brockus CW, Ackermann MR, Thomas P, Meng XJ, Halbur PG. Effect of Vaccination with selective Bacterins on conventional Pigs infected with Type 2 Porcine Circovirus. Vet Pathol 2003; 40(6):521–529.

    Article  PubMed  CAS  Google Scholar 

  15. Opriessnig T, Fenaux M, Yu S, Evans RB, Cavanaugh D, Gallup JM, Ackermann MR, Pallares FJ, Thacker EL, Lager KM, Meng XJ, Halbur PG. Effect of Porcine Parvovirus Vaccination on the Development of PMWS in Segregated Early Weaned Pigs Coinfected with Type 2 Porcine Circovirus and Porcine Parvovirus. Veterinary Microbiology 2004; 98(3–4):209–220.

    Article  PubMed  CAS  Google Scholar 

  16. Meyerholz DK, Gallup JM, Grubor BM, Evans RB, Tack BF, McCray PB Jr., Ackermann MR. Developmental expression and distribution of sheep b-defensin-2. Dev Comp Immun 2004; 28:171–178.

    Article  CAS  Google Scholar 

  17. Grubor BM, Gallup JM, Meyerholz DK, Crouch EC, Evans RB, Brogden KA, Lehmkuhl HD, Ackermann MR. Enhanced Surfactant Protein and Defensin mRNA Levels and Reduced Viral Replication during Parainfluenza Virus Type 3 Pneumonia in Neonatal Lambs. CDLI 2004; 11(3):599–607.

    PubMed  CAS  Google Scholar 

  18. Ackermann MR, Gallup JM, Zabner J, Evans RB, Brockus CW, Meyerholz DK, Grubor B, Brogden KA. Differential expression of sheep beta-defensin-1 and - 2 and interleukin 8 during acute Mannheimia haemolytica pneumonia. Microbial Pathogenesis 2004; 37:21–27.

    Article  PubMed  CAS  Google Scholar 

  19. Grubor B, Gallup JM, Ramírez-Romero R, Bailey TB, Crouch EC, Brogden KA, Ackermann MR. Surfactant Protein D expression in normal and pneumonic ovine lung. Vet Immunology & Immunopathology 2004; 101:235–242.

    Article  CAS  Google Scholar 

  20. Meyerholz DK, Grubor B, Fach SJ, Sacco RE, Lehmkuhl HD, Gallup JM, Ackermann MR. Reduced Clearance of Respiratory Syncytial Virus in a Preterm Lamb Model. Microbes & Infection 2004; 6(14):1312–1319.

    Article  Google Scholar 

  21. Meyerholz DK, Grubor B, Gallup JM, Lehmkuhl HD, Anderson RD, Lazic T, Ackermann MR. Adenovirus-Mediated Gene Therapy Enhances Parainfluenza Virus 3 Infection in Neonatal Lambs. J Clin Micro 2004; 42(10):4780–4787.

    Article  CAS  Google Scholar 

  22. Broackes-Carter F, Mouchel N, Gill D, Hyde S, Bassett J, Harris A. Quantitative CFTR transcript analysis by TaqMan. Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy. Hum Mol Genet 2002; 11:125–131.

    Article  PubMed  CAS  Google Scholar 

  23. Swillens S, Goffard J-C, Marechal Y, de Kerchove d’Exaerde A, Housni EH. Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve. Nuc Acids Res 2004; 32(6):e53.

    Google Scholar 

  24. Peirson SN, Butler JN, Foster RG. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nuc Acids Res 2003; 31(14):e73.

    Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Jack M. Gallup.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Gallup, J.M., Kawashima, K., Lucero, G. et al. New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR. Biol. Proced. Online 7, 70–92 (2005).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI:

Indexing terms