LETTER TO THE EDITOREleven Golden Rules of Quantitative RT-PCRReverse transcription followed by quanti-tative polymerase chain reaction analysis,or qRT-PCR, is an extremely sensitive,cost-effective method for quantifying genetranscripts from plant cells. The availabil-ity of nonspecific double-stranded DNA(dsDNA) binding fluorophors, such asSYBR Green, and 384-well-plate real-timePCR machines that can measure fluores-cence at the end of each PCR cycle make itpossible to perform qRT-PCR on hundredsof genes or treatments in parallel. This hasfacilitated the comparative analysis of allmembers of large gene families, such astranscription factor genes (Czechowskiet al., 2004). Given the relatively low costof PCR reagents, and the precision, sensi-tivity, flexibility, and scalability of qRT-PCR,it is little wonder that thousands of researchlabs around the world have embraced it asthe method of choice for measuring tran-script levels. However, despite its popular-ity, we continue to see systematic errors inthe application of methods for qRT-PCRanalysis, which can compromise the inter-pretation of results. The letter to the editorby Gutierrez et al. in this issue highlightsone of many common sources of error,namely, the inappropriate choice of refer-ence genes for normalizing transcript levelsof test genes prior to comparative analysisof different biological samples. The follow-ing are 11 golden rules of qRT-PCR that,when observed, should ensure reproduc-ible and accurate measurements of tran-script abundance in plant and other cells.These rules are for relative quantification ofRNA using two-step RT-PCR (where theproduct of a single RT reaction is used astemplate in multiple PCR reactions), SYBRGreen to detect gene-specific PCR prod-ucts, and reference genes for normalizingtranscript levels of test genes before com-paring samples. Further details can befound elsewhere (Czechowski et al., 2004,2005). Most of these rules also apply torelative quantification methods that em-ploy sequence-specific fluorescent probes,such as TaqMan probes, and to absolutequantification methods (http://www.gene-quantification.info/).(1) Harvest material from at least threebiological replicates to facilitate statisticalanalysis of data, freeze immediately inliquid nitrogen, and store at 280Ctopreserve full-length RNA.(2) Use an RNA isolation procedure thatproduces high-quality total RNA from allsamples to be analyzed. Check RNA qual-ity using an Agilent 2100 Bioanalyzer (RNAintegrity number, RIN . 7 and ideally . 9)or by electrophoretic separation on a high-resolution agarose gel (look for sharpethidium bromide–stained rRNA bands)and spectrophotometry (A260/A280. 1.8and A260/A230. 2.0). Quantify RNA usingA260values.(3) Digest purified RNA with DNase I toremove contaminating genomic DNA,which can act as template during PCRand lead to spurious results. Subsequently,perform PCR on the treated RNA, usinggene-specific primers, to confirm absenceof genomic DNA.(4) Perform RT reactions with a robust re-verse transcriptase with no RNaseH activity(like SuperScriptIII from Invitrogen or Array-Script from Ambion) to maximize cDNAlength and yield. Use ultraclean oligo(dT)primer of high integrity. qRT-PCR geneexpression measurements are comparableonly when the same priming strategy andreaction conditions are used in all experi-ments and reactions contain the same totalamount of RNA (Sta˚hlberg et al., 2004).(5) Test cDNA yield and quality. PerformqPCR on an aliquot of cDNA from eachsample, using primers to one or more ref-erence genes that are known to be stablyexpressed in the organ(s)/tissue(s) underthe range of experimental conditions tested.Threshold cycle (Ct) values should be withinthe range mean 61 for each reference geneacross all samples to ensure similar cDNAyield from each RT reaction. Quality ofcDNA can be assessed using two pairs ofprimers for a reference gene that are ;1kbapart. Typically, the Ct value for the primerpair at the 5#-end of a cDNA will be higherthan the Ct value of the primer pair at the3#-end, as reverse transcription begins atthe 3# [poly(A)] end of the template mRNAand does not always extend to the 5#-end ofthe template. Ideally, the Ct value of the5#-end primer pair should not exceed that ofthe 3#-end pair by more than one cyclenumber.(6) Design gene-specific PCR primersusing a standard set of design criteria (e.g.,primer Tm¼ 60 6 1C, length 18 to 25bases, GC content between 40 and 60%),which generate a unique, short PCR prod-uct (between 60 and 150 bp) of the ex-pected length and sequence from a complexcDNA sample in preliminary tests, to facili-tate multiparallel qPCR using a standardPCR program. The 3#-untranslated region isa good target for primer design because itis generally more unique than coding se-quence and closer to the RT start site.(7) Reduce technical errors in PCR re-action setup by standardizing (robotize ifpossible) and minimizing the number ofpipetting steps. Mix cDNA with qPCRreagents, then aliquot a standard volumeof this ‘‘master mix’’ into each reaction wellcontaining a standard volume of specificprimers. Set up reactions in a clean envi-ronment free of dust, preferably under apositive airflow hood. Routinely check forDNA contamination of primer and reagentstocks by performing PCR reactions on notemplate (water) controls.(8) For relative quantification of transcriptlevels, design and test gene-specificprimers for at least four potential referencegenes selected from the literature (e.g.,Czechowski et al., 2005) or from your ownexperience that are likely to be stablyexpressed throughout all organs and treat-ments to be compared. Validate referencegenes in preliminary experiments on therange of tissues and treatments you wishto compare using a foreign cRNA added toeach RNA sample prior to RT-PCR towww.plantcell.org/cgi/doi/10.1105/tpc.108.061143The Plant Cell, Vol. 20: 1736–1737, July 2008, www.plantcell.org ª 2008 American Society of Plant Biologistsnormalize data for reference gene tran-scripts prior to assessment of their expres-sion stability (Czechowski et al., 2005).(9) Perform real-time PCR on test andreference genes in parallel for each sampleto capture fluorescence data on dsDNAafter each cycle of amplification. Also,perform dsDNA melting curve analysis atthe end of the PCR run. When relying onnonspecific DNA binding fluorophors, suchas SYBR Green, to quantify relative dsDNAamount, ensure that only a single PCRamplicon of the expected