APPROACHES TO MESSENGER RNA DETECTION - ResearchGate

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134 Z. Dvořák, J.-M. Pascussi, M. ModrianskýSWOT: Strength: Owing to its unique principle, themethod gives nearly absolute specificity. It is therefore themost specific from the three methods under review 6 . Consequently,the design of the probe is intended to recognizea specific sequence like in other techniques. Compared tothe NB, RPA is approximately ten times more sensitive.Weakness: The method is time consuming and requiresa highly skilled operator. The work organisation is verytiresome for the operator because there is no chance forpostponing any of the steps after hybridization. Furthermore,economical expenses are higher as compared toNB. Opportunity: Due to its high specificity, two or severalgenes with high homology in primary structure can be distinguished.Recall again that only one base pair mismatchleads to the cleavage by RNAses. Threat: Since the procedureconsists of many steps, the risk of RNA degradationin sample during the analysis is very high and occursquite frequently. The risk of radioactivity contaminationwhen handling samples and particularly during riboprobepreparation is also considerable, even though the activitiesused are much lower than in the NB method.REAL TIME POLYMERASE CHAIN REACTIONThanks to its sensitivity even very low abundancemRNA can be detected using this technique. Other frequentuses include: primary validation of gene expressionand hence confirmation of microarray data, splice variantanalysis, allelic discrimination, viral titre (e.g. HCV),bacterial species identification, and single nucleotidepolymorphism genotyping.Method description: This method takes an advantagefrom well known polymerase chain reaction (PCR)which is used for DNA detection 7 . The abbreviation RT-is somewhat ambiguous standing on one hand for reversetranscription reaction, a key step in the procedure, and onthe other hand for real time format of the analysis whenLightcycler apparatus is used 8 .First of all, the reverse transcription reaction is performedusing RNA-dependent DNA polymerase (reversetranscriptase), synthesising the cDNAs complementaryto the mRNAs present in the sample. It is sine qua nonto use sample of RNA in water free of organic solventsresidues, e.g. phenol, isopropanol, chloroform, ethanoletc., to avoid inhibition of reverse transcriptase. Forthe starting material it is possible to use total RNA, atleast 1 µg, but isolated mRNA is more appropriate givinghigher probability of low copy mRNA transcription.The synthesised cDNA is mixed with DNA-dependentDNA polymerase, sufficient amount of deoxynucleotidetriphosphates (dATP, dCTP, dGTP, and dTTP), andfluorescently labeled oligonucleotide primers. As in thecase of “classical” PCR, one cycle of a PCR reactionconsists of strand separation, annealing, and elongationsteps. Duration of and temperature during each of thethree steps are set on the thermocycler apparatus in use.Fluorescence is monitored in each sample following everycycle of the PCR reaction. When threshold fluorescenceintensity is reached due to increased number of copies ofthe cDNA of interest, the fluorescence linearly increasesin each Asubsequent cycle eventually reaching a maximumplateau. The threshold RIF (M) is so-called Actin“crossing CYP3A4 point” whichis essential for comparison between samples. Computersoftware provided0with the Lightcycler14.69usually27.84calculatesthe cycle number 1corresponding 14.86to the crossing 22.94 point usingsecond derivative 5 maximum. 15.07If calibrated 22.24for knownnumber of cDNA copies present in a sample, RT-PCR canbe used for very precise 10 quantification 14.78 of 21.56 original mRNAcopies per number 20 of cells 8, 15.319 . Representative 21.52computeroutput from a Lightcycler apparatus is shown in Fig 3.ARIF (M)0151020Actin14.6914.8615.0714.7815.31CYP3A427.8422.9422.2421.5621.52BFlod inductionnormalized per actinInduction of CYP3A4 by rifampicin1501005000 1 5 10 20Rifampicin (M)Fig. 3. Example of Real-Time Polymerase Chain Reaction analysis. RT-PCR computer data from total RNA isolated fromB primary Induction human hepatocytes of CYP3A4 by using rifampicin Trizol procedure. Panel A) shows cycle thresholds for CYP3A4 and actin mRNA.Panel B) 150shows fold induction of CYP3A4 gene by rifampicin. Data are normalized per actin value.Flod inductionnormalized per actin1005000 1 5 10 20Rifampicin (M)
Approaches to messenger RNA detection – comparison of methods135SWOT: Strength: Method is elegant, safe, simple touse, and requires only minimum experience and skills.It provides maximum sensitivity of all three discussedmethods (approx. 10 000 times more sensitive than NB)and among them it is the only one allowing quantification.Weakness: This method is the most expensive: Lightcyclerapparatus, fluorescently labeled primers, and mRNAisolation kits are the principal costs. There is a need ofhighly pure samples to be used, free of organic solvents,often present after Trizol isolation increasing the needfor alternative isolation procedures. Opportunity: Owingto its high sensitivity, a large number of genes can beanalysed from a single mRNA preparation. Threat: Theselection and design of the primers must be done carefullybecause of the possibility, albeit minuscule, thatother homologous genes may be detected. The risk ofRNA degradation, until the reverse transcription stepis performed, is high because of water used as solvent.Contamination of RNA by fragments of genomic DNAcan occur if Trizol is used for extraction. Consequently, itcannot be distinguished whether genomic DNA or reversetranscript are detected.CONCLUSIONWith the human genome now available a principalquestion was raised: what do all those genes do? In view ofmany a researcher this question translates primarily intothe problem of: Are all those genes transcribed? It meansto look for mRNA occurrence within a cell as many ofthe genes may be cell type specific. Area of genomics isnot the only one with craving for mRNA data. The threemethods described herein are the tools currently availablefor a researcher to deal with a particular problem involvingmRNA.We offer a SWOT analysis in attempt to help othersduring pros and cons consideration when selectinga method for their purpose. Things to consider areobviously intertwined as specificity of each method islinked to financial demands together with health and environmentalrisks. An ideal of a highly specific, safe, andinexpensive method for mRNA detection does not exist.Quite often combination of two, out of the three methodsdiscussed, is utilized in many a lab throughout the world.This is important especially in pilot studies in order toconfirm data obtained. Therefore we recommend to haveone of these methods for routine use as the core methodand then to select another one not as an alternative butas a confirmation tool.ACKNOWLEDGEMENTThe authors gratefully acknowledge financial supportfrom the Ministry of Education of the Czech Republic grantMSM 151100003.REFERENCES1. Chomczynski P, Sacchi N. (1993) Single-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 162, 156–9.2. Kingston RE. (1993) Preparation of Poly(A) + RNA. In: CurrentProtocols in Molecular Biology (Eds.: Ausubel EM, Brent R,Kingston RE, Moore DD, Siedman JG, Smith JA, Struhl K),p 4.5.1.–4.5.3. John Wiley and Sons, New York, NY, 1993.3. Brown T. (1993) Analysis of RNA by Northern and Slot BlotHybridization. In: Current Protocols in Molecular Biology (Eds.:Ausubel EM, Brent R, Kingston RE, Moore DD, Siedman JG,Smith JA, Struhl K), p 4.9.1.–4.9.14. John Wiley and Sons, NewYork, NY, 1993.4. Gilman M. (1993) Ribonuclease protection assay. In: CurrentProtocols in Molecular Biology (Eds.: Ausubel EM, Brent R,Kingston RE, Moore DD, Siedman JG, Smith JA, Struhl K),p 4.7.1.–4.7.8. John Wiley and Sons, New York, NY, 1993.5. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, GreenMR. (1984) Efficient in vitro synthesis of biologically activeRNA and RNA hybridization probes from plasmids containinga bacteriophage SP6 promoter. Nucleic Acids Res 12, 7035–56.6. Rottman JB. (2002) The ribonuclease protection assay: a powerfultool for the veterinary pathologist. Vet Pathol 39, 2–9.7. Erlich HA. (ed.) (1989) PCR Technology: Principles and Applicationsfor DNA amplification. Stockton Press, London.8. Wilkening S, Bader A. (2003) Influence of culture time on theexpression of drug-metabolizing enzymes in primary human hepatocytesand hepatoma cell line HepG2. J Biochem Mol Toxicol 17,207–13.9. Wilkening S, Stahl F, Bader A. (2003) Comparison of primaryhuman hepatocytes and hepatoma cell line HepG2 with regardto their biotransformation properties. Drug Metab Dispos 31,1035–42.
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