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Analysis of mRNA Levels by RT-PCR 414Quantitative Analysis of mRNA Levelsin Xenopus Embryos by ReverseTranscriptase–Polymerase Chain Reaction (RT-PCR)Oliver C. Steinbach and Ralph A. W. Rupp1. IntroductionOver the last few years, RT-PCR (1,2) has become a widely accepted methodfor quantitation of steady-state mRNA levels, particularly in Xenopus. Itsunmatched sensitivity and swiftness allows for a high sample throughput withminimal amounts of starting material—considerable advantages over the conventionalmethods of Northern blotting or RNase protection. Initially, the useof RT-PCR for quantitative analysis was viewed skeptically. This was basedon the concern that minor differences in the reaction conditions betweensamples would erratically influence the exponential rate of PCR amplification;therefore, results would be skewed a priori. This theoretical concern has turnedout to be irrelevant for most applications.Here, we describe a basic protocol for RT-PCR analysis, which has beenoptimized in our laboratory. In the first step, total cellular RNA is reversetranscribedinto a random-primed, first-strand cDNA library. This library isthen used as the template for PCR reactions, in which gene-specific primersamplify short regions from the respective cDNAs. The PCR products are tracelabeled with radioisotopes during synthesis and thereby can be easily detectedand quantitated after size fractionation by polyacrylamide gel electrophoresis.In its routine application, this method allows one to determine relative mRNAlevels by using reference mRNAs for normalization. Ideally, test and referencecDNA templates are coamplified in the same tube (“multiplex” PCR). Ifrequired, absolute transcript numbers can also be obtained by a competitivePCR assay (3). In general, our method requires less than 0.5 µg of total cellularFrom: Methods in Molecular Biology, Vol. 127: Molecular Methods in Developmental Biology:Xenopus and Zebrafish Edited by: M. Guille © Humana Press Inc., Totowa, NJ41
42 Steinbach and RuppRNA per sample, measures reliably more than 100-fold differences in relativetranscript abundance, and detects as little as 100 mRNA molecules; raw datafrom 50 or more samples can be obtained within 48 h.Messenger RNA quantitation by RT-PCR strictly requires that PCR productamounts be directly proportional to the original mRNA template amounts.Establishing experimental conditions, which ensure a quantitative performanceof RT-PCR, is usually thought to be a major obstacle. Many different parametersinfluence the performance of the assay, and conditions have to be establishedfor each primer pair. Therefore, we have included in this chapter anelaborate guide for calibration of RT-PCR conditions, which has been evolvedfrom practical experience. By no means does this guide claim to be exclusive,although in a few cases we felt obliged to give absolute values for criticalparameters. We hope that it will help the reader save time and effort.2. Materials2.1. General Materials and Equipment1. Automated thermal cycler.2. Benchcoat.3. Deionized (Millipore quality), autoclaved water (dH 2 O).4. Diethyl pyrocarbonate-treated H 2 O (DEPC–H 2 O). Caution: DEPC is extremelytoxic.5. 0.5 mL PCR tubes, 1.5- and 2-mL microfuge tubes.6. Gloves.7. Heating block (variable temperature).8. Micropipet (two sets): 0.1–2 µL, 2–20 µL, 10–200 µL, 100–1000 µL, and appropriatetips.9. Tabletop microcentrifuges with fixed-angle rotors at room temperature and 4°C.2.2. RNA Purification1. Chloroform (ACS grade), store at room temperature. Caution: chloroform isan irritant.2. 70% Ethanol (ACS grade) in DEPC–H 2 O, store at –20°C.3. Isopropanol/tRNA: 60 µg/mL tRNA in 100% isopropanol (ACS grade), store at 4°C.Dilute the Escherichia coli tRNA (e.g., from strain MRE600, Roche Diagnostics,Mannheim, Germany, see Note 1) from a 10 mg/mL stock in DEPC–H 2 O, store at –20°.4. RNA isolation solvent for single step separation (e.g., TriStar, AGS, Heidelberg).(Caution: The solvent contains phenol and is toxic.)2.3. RT Reaction1. RT–dNTP mix: 2.5 mM each dCTP, dGTP, dATP, dTTP diluted in DEPC–H 2 O,store at –20°C.2. 0.1 M dithiothreitol (DTT) in DEPC–H 2 O, store at –20°C.
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