Reverse Transcription

All normal cells in a human's body, with few exceptions, have the same chromosomal DNA sequence, that is, the same genetic code. Thus, genomic information obtained from the DNA of easily obtained normal white blood cells would be applicable to the genetic makeup of normal lung, brain, or colon cells. This idea does not apply to malignant cells, which can have a genetic composition that is profoundly different from that of normal cells.

Function and structure of various cell types differ because of the mRNA that they transcribe and ultimately the proteins that are translated. In other words, it is the protein expression profile of cells that differentiates them. Presently, the most practical way to study the specific genes expressed in a particular cell type is to analyze the mRNA the cells make.

Because RNA is unstable and therefore difficult to work with in the laboratory, it can be converted into the complementary DNA (cDNA) by a process known as reverse transcrip-tion.69-71 The resultant cDNA is much more stable than the mRNA. Reverse transcription is so named because RNA is used as the template to direct the production of DNA: the reverse of normal cellular transcription, where DNA is used by RNA polymerase to direct the production of mRNA.

A reverse transcriptase enzyme is an RNA-directed DNA polymerase made and used by some RNA viruses to complete their life cycle within a host. Viral reverse transcriptases have been characterized and/or cloned, and the enzymes are commercially available for use in research and clinical molecular laboratories.72-76

Reverse transcriptase, similar to DNA polymerase, requires a DNA primer (Fig. 9.5) to initiate its function. Because mRNA has a poly(A) tail at its 3' 'end, an ideal primer for reverse transcription of mRNA species would be a poly(T) oligonucleotide (oligo dT).77,78 A replete collection of short DNA primers with random sequences can also be used; these primers are recommended if reverse transcription of ribosomal RNA (rRNA) is also desired along with mRNA. The enzyme starts transcription at the 3'-end of template RNA [the 5' -end of the nascent (new) cDNA strand] and proceeds in a 5'—> 3'-direction on the nascent strand ("first strand synthesis"). In this fashion, all the mRNA (or total RNA) present in a cell can be transcribed into complementary DNA. Those mRNA sequences that are present at a high copy number in the cell will be reverse transcribed to a high cDNA copy number compared with those mRNA sequences which are rare in a cell.

A typical reverse transcription protocol is given in Table 9.3. The two most commonly used reverse transcriptases are from bird and mouse viruses: avian myeloblastosis virus

Reverse Transcriptase
  1. 9.5. Reverse transcription. Reverse transcriptase uses oligo dT as the primer on the target mRNA and polymerizes in the 5'—> 3'-direction on the new DNA strand. The original mRNA strand is then cleaved by an RNAse domain within the reverse transcriptase (not shown), thus allowing polymerization of the single-stranded DNA into double-stranded DNA during poly-merase chain reaction .
  2. 9.5. Reverse transcription. Reverse transcriptase uses oligo dT as the primer on the target mRNA and polymerizes in the 5'—> 3'-direction on the new DNA strand. The original mRNA strand is then cleaved by an RNAse domain within the reverse transcriptase (not shown), thus allowing polymerization of the single-stranded DNA into double-stranded DNA during poly-merase chain reaction .

Table 9.3. Typical reverse transcription reaction protocol.

RNA 1-2 mg DEPC-treated H2O

Oligo dT or random primers at 40 mM 70°C I 5min 4°C I 5min

Quickly add 20.0 ml of prepared RT master mix

DEPC-treated H2O: add to total final volume

MMLV (10x) or AMV (5x) buffer dNTP at 10 mmole

RNAse inhibitor 10-40 units

MMLV reverse transcriptase 200 unitsa

AMV reverse transcriptase 30 unitsa

Use ~1-3 ml in the PCR reaction

  1. 0 ml 8.5 ml 2.0 ml
  2. 0 ml 2.0-4.0 ml 3.0 ml 1.0 ml

AMV avian myeloblastosis virus; DEPC diethylpyrocarbonate; dNTP deoxynucleotide triphosphate; MMLV Moloney murine leukemia virus; PCR polymerase chain reaction; RT reverse transcription aOne unit of reverse transcriptase is defined as the amount of enzyme that will incorporate 1 nmole of deoxythymidine triphosphate into acid-insoluble material in 10 min at 37°C using poly(rA), oligo(dT) as template primer

(AMV) and Moloney murine leukemia virus (MMLV).79 Their recommended buffers should not be interchanged. RNAse inhibitors and diethylpyrocarbonate-treated water are needed to preserve the unstable RNA. The initial 70°C heating is to remove secondary structures from the RNA; the 42°C (AMV and some MMLV products) or 37°C (some MMLV products) incubations are the working temperature of the enzymes. The 90-95°C step is needed to inactivate the enzymes.

The cDNA made by reverse transcription of mRNA (and/ or rRNA) can then be used as a template for PCR if the appropriate primers for the target DNA are present. During the first cycle of the PCR, only one (the forward) primer is needed because only one strand is polymerized, but this new strand will serve as the template for the opposite primer during the second PCR cycle, and polymerization of both strands will continue with each cycle. [Note that some bacteria such as Thermus thermophilus have an enzyme (Tth) that can both reverse transcribe RNA and polymerize DNA, allowing reverse transcription and PCR to proceed simultaneously in a single tube.]

Reverse transcription-PCR (RT-PCR) is thus an important tool that allows the investigator to study the genes expressed or not expressed in specific cells after isolation of the mRNA.80-83 Additional (post-PCR) techniques such as gel electrophoresis, single-stranded conformation polymorphism gels, restriction fragment length polymorphism analysis, DNA sequencing, microarrays, and so forth, can be applied to also determine if the genes expressed have mutations.

Under- or overexpression of a particular gene in neoplastic or reactive cells can be investigated by comparing their expression levels in normal cells, which could be done by comparing band strengths on Northern (RNA) blots. However, in these methods one must control for the number of tumor/reactive cells being the same as the number of normal cells. Analysis is much easier if done by real-time PCR, in which the ratio of the expression level of the gene of interest is compared with the expression level of a constitutively expressed housekeeping gene such as ß-actin, 18S rRNA, cyclophilin, glyceraldehyde-3-phosphate dehydrogenase, and ß2-microglobulin. This ratio is calculated in both the normal and neoplastic/reactive cells, and then the ratios are compared to see if there is relative up-or downregulation of the gene of interest.

References

  1. Bell J. The polymerase chain reaction. Immunol Today. 1989; 10: 351-355.
  2. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230:1350-1354.
  3. Mullis K, Faloona F, Scharf S, et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(pt 1):263-273.
  4. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA poly-merase. Science. 1988;239:487-491.
  5. Saboor SA, Johnson NM, McFadden J. Detection of mycobac-terial DNA in sarcoidosis and tuberculosis with polymerase chain reaction. Lancet. 1992;339:1012-1015.
  6. Myerson D, Lingenfelter PA, Gleaves CA, et al. Diagnosis of cytomegalovirus pneumonia by the polymerase chain reaction with archived frozen lung tissue and bronchoalveolar lavage fluid. Am J Clin Pathol. 1993;100:407-413.
  7. Raad I, Hanna H, Huaringa A, et al. Diagnosis of invasive pulmonary aspergillosis using polymerase chain reaction-based detection of aspergillus in BAL. Chest. 2002;121:1171-1176.
  8. Sundaresan S, Alevy YG, Steward N, et al. Cytokine gene transcripts for tumor necrosis factor-alpha, interleukin-2, and inter-feron-gamma in human pulmonary allografts . J Heart Lung Transplant. 1995;14:512-518.
  9. Lordan JL, Bucchieri F, Richter A, et al. Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells. J Immunol. 2002;169:407-414.
  10. Nogee LM, Dunbar AE III, Wert SE, et al. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. NEnglJMed. 2001;344:573-579.
  11. Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas . J Mol Diagn. 2005;7:396-403.
  12. Westra WH, Baas IO, Hruban RH, et al. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res. 1996;56:2224-2228.
  13. Pulte D , Li E , Crawford BK , et al. Sentinel lymph node mapping and molecular staging in nonsmall cell lung carcinoma. Cancer. 2005;104:1453-1461.
  14. Bohlmeyer T, Le TN , Shroyer AL , et al. Detection of human papillomavirus in squamous cell carcinomas of the lung by polymerase chain reaction. Am J Respir Cell Mol Biol. 1998;18:265-269.
  15. Bremnes RM , Sirera R , Camps C . Circulating tumour-derived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up? Lung Cancer. 2005;49:1-12.
  16. Eisenstein BI . The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N Engl J Med. 1990;322:178-183.
  17. Wenham PR . DNA-based techniques in clinical biochemistry: a beginner's guide to theory and practice . Ann Clin Biochem. 1992;29(pt 6):598-624.
  18. Remick DG, Kunkel SL, Holbrook EA, Hanson CA. Theory and applications of the polymerase chain reaction. Am J Clin Pathol. 1990;93:S49-S54.
  19. Chakrabarti R, Schutt CE. The enhancement of PCR amplification by low molecular-weight sulfones. Gene. 2001;274:293-298.
  20. Laksanalamai P, Pavlov AR, Slesarev AI, Robb FT. Stabilization of Taq DNA polymerase at high temperature by protein folding pathways from a hyperthermophilic archaeon, Pyrococ-cus furiosus. Biotechnol Bioeng. 2006;93:1-5.
  21. Breslauer KJ, Frank R, Blocker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986;83:3746-3750.
  22. SantaLucia J Jr, Allawi HT, Seneviratne PA. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry. 1996;35:3555-3562.
  23. Wallace RB, Shaffer J, Murphy RF, et al. Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 1979;6:3543-3557.
  24. Chien A , Edgar DB , Trela JM . Deoxyribonucleic acid poly-merase from the extreme thermophile Thermus aquaticus. J Bacteriol. 1976;127:1550-1557.
  25. Takagi M, Nishioka M, Kakihara H, et al. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol. 1997;63:4504-4510.
  26. Davidson JF, Fox R , Harris DD , et al . Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the proces-sivity and fidelity of Taq DNA polymerase. Nucleic Acids Res. 2003;31:4702-4709.
  27. Keohavong P, Thilly WG. Fidelity of DNA polymerases in DNA amplification. ProcNatlAcadSciUSA 1989;86:9253-9257.
  28. Wilson IG . Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol. 1997;63:3741-2751.
  29. Khan G, Kangro HO, Coates PJ, Heath RB. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol. 1991;44:360-365.
  30. Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI. Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends Biotechnol. 2004;22:253-260.
  31. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128.
  32. Eckert KA, Kunkel TA. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990;18:3739-3744.
  33. McPherson MJ, M0ller SG. PCR. Oxford, UK: BIOS Scientific ; 2000 .
  34. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 ;98: 503-517.
  35. Bevan IS, Rapley R, Walker MR. Sequencing of PCR-amplified DNA. PCR Methods Appl. 1992;1:222-228.
  36. Moretti T, Koons B, Budowle B. Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA poly-merase. Biotechniques. 1998;25:716-722.
  37. Brandwein M, Zeitlin J, Nuovo GJ, et al. HPV detection using "hot start" polymerase chain reaction in patients with oral cancer: a clinicopathological study of 64 patients. Mod Pathol. 1994;7: 720-727.
  38. Nuovo GJ, Gallery F, MacConnell P. Detection of amplified HPV 6 and 11 DNA in vulvar lesions by hot start PCR in situ hybridization. Mod Pathol. 1992;5:444-448.
  39. Chou Q, Russell M, Birch DE, et al. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications . Nucleic Acids Res. 1992 ; 20 : 1717-1723.
  40. Tilston P, Corbitt G . A single tube nested PCR for the detection of hepatitis C virus RNA. J Virol Methods. 1995;53:121-129.
  41. Smit VT, Boot AJ, Smits AM, et al. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res. 1988;16:7773-7782.
  42. Herman JG , Graff JR , Myohanen S , et al . Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:9821-9826.
  43. Curtis CD, Goggins M. DNA methylation analysis in human cancer. Methods Mol Med. 2005;103:123-136.
  44. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18:1427-1431.
  45. Bird A. The essentials of DNA methylation. Cell. 1992;70:5-8.
  46. Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis. 2000;21:461-467.
  47. RheeI,BachmanKE,ParkBH,etal.DNMT1 andDNMT3bcoop-erate to silence genes in human cancer cells. Nature. 2002;416: 552 - 556 .
  48. Dammann R , Takahashi T, Pfeifer GP. The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene. 2001;20:3563-3567.
  49. Zochbauer-Muller S, Fong KM, Virmani AK, et al. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res. 2001;61:249-255.
  50. Clark SJ , Harrison J , Paul CL , Frommer M . High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994 ; 22 : 2990 - 2997 .
  51. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev. 2000;13:559-570.
  52. Richards B , Skoletsky J , Shuber AP, et al . Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet. 1993;2:159-163.
  53. Scurto P, Hsu Rocha M, Kane JR, et al. A multiplex RT-PCR assay for the detection of chimeric transcripts encoded by the risk-stratifying translocations of pediatric acute lymphoblastic leukemia. Leukemia. 1998;12:1994-2005.
  54. Pallisgaard N, Hokland P, Riishoj DC, et al. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood. 1998;92:574-588.
  55. Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). NucleicAcidsRes. 1989;17:2503-2516.
  56. Saiki RK, Bugawan TL, Horn GT, et al. Analysis of enzymati-cally amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature. 1986;324:163-166.
  57. Schaefer BC. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem. 1995;227:255-273.
  58. Komminoth P, Long AA . In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch B Cell Pathol Incl Mol Pathol. 1993;64:67-73.
  59. Livak KJ , Flood SJ , Marmaro J , et al . Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCRMethodsAppl. 1995;4:357-362.
  60. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-994.
  61. Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5' nuclease assays. Curr Opin Biotechnol. 1998;9:43-48.
  62. Holland PM , Abramson RD , Watson R , Gelfand DH . Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. ProcNatlAcadSciUSA. 1991;88:7276-7280.
  63. Longley MJ, Bennett SE, Mosbaugh DW. Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990;18:7317-7322.
  64. Förster T. Zwischemolekulare energiewanderung und fluoreszenz. Ann Physik. 1948;2:55-67.
  65. Stryer L, Haugland RP. Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A. 1967;58:719-726.
  66. Grinvald A, Haas E, Steinberg IZ. Evaluation of the distribution of distances between energy donors and acceptors by fluorescence decay. Proc Natl Acad Sci U S A. 1972;69:2273-2277.
  67. Oliver DH , Thompson RE , Griffin CA , Eshleman JR . Use of single nucleotide polymorphisms (SNP) and real-time poly-merase chain reaction for bone marrow engraftment analysis. J Mol Diagn. 2000;2:202-208.
  68. Lay MJ , Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem. 1997 ; 43 : 2262 - 2267 .
  69. Temin HM , Mizutani S . RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970;226:1211-1213.
  70. Baltimore D . RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970;226:1209-1211.
  71. Spiegelman S, Burny A, Das MR, et al. Characterization of the products of DNA-directed DNA polymerases in oncogenic RNA viruses. Nature. 1970;227:563-567.
  72. Shinnick TM, Lerner RA, Sutcliffe JG. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981;293:543-548.
  73. Reddy EP, Smith MJ, Aaronson SA. Complete nucleotide sequence and organization of the Moloney murine sarcoma virus genome. Science. 1981;214:445-450.
  74. Kotewicz ML, D'Alessio JM, Driftmier KM, et al. Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene. 1985;35:249-258.
  75. Verma IM, Baltimore D. Purification of the RNA-directed DNA polymerase from avian myeloblastosis virus and its assay with polynucleotide templates. Methods Enzymol. 1974;29:125-130.
  76. Houts GE, Miyagi M, Ellis C, et al. Reverse transcriptase from avian myeloblastosis virus. J Virol. 1979;29:517-522.
  77. Verma IM. Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H . J Virol. 1975;15:843-854.
  78. Marcus SL, Modak MJ. Observations on template-specific conditions for DNA synthesis by avian myeloblastosis virus DNA polymerase. NucleicAcidsRes. 1976;3:1473-1486.
  79. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001:A4.24.
  80. Broackes-Carter FC, Mouchel N, Gill D, et al. Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy. Hum Mol Genet. 2002;11:125-131.
  81. Dagnon K, Pacary E, Commo F, et al. Expression of erythropoi-etin and erythropoietin receptor in non-small cell lung carcinomas. Clin CancerRes. 2005;11:993-999.
  82. Singhal S , Wiewrodt R , Malden LD , et al . Gene expression profiling of malignant mesothelioma. Clin Cancer Res. 2003 ; 9 : 3080 - 3097 .
  83. Lam KM, Oldenburg N, Khan MA, et al. Significance of reverse transcription polymerase chain reaction in the detection of human cytomegalovirus gene transcripts in thoracic organ transplant recipients. J Heart Lung Transplant. 1998;17:555-565.
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