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To accurately evaluate expression levels of target genes, stable internal reference genes is required for normalization of quantitative real-time PCR (qRT-PCR) data. However, there have been no systematical investigation on the stability of reference genes used in the bedstraw weed,  Galium aparine L . (BGA). In this study, the expression profiles of seven traditionally used reference genes, namely  18S ,  28S ,  ACT ,  GAPDH ,  EF1α ,  RPL7  and  TBP  in BGA were assessed under both biotic (developmental time and tissue), and abiotic (temperature, regions and herbicide) conditions. Four analytical algorithms (geNorm, Normfinder, BestKeeper and the ΔCt method) were used to analyze the suitability of these genes as internal reference genes. RefFinder, a comprehensive analytical software, was used to rank the overall stability of the candidate genes. The optimal normalization internal control genes were ranked as:  28S  and  RPL7  were best for all the different experimental conditions (developmental stages, tissues, temperature, regions and herbicide treatment);  28S  and  RPL7  for developmental stages;  TBP  and  GAPDH  for different tissues;  28S  and  GAPDH  were relatively stable for different temperature;  28S  and  TBP  were suitable for herbicide treatment. A specific set of reference genes were recommended for each experimental condition in BGA.

Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L.