Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 of miRNA mediated gene regulation in development and disease such as cancer or viral infections was recognised very early. However, very recent studies suggest that miRNAs are also involved in the specific host response to bacterial pathogens such as Mycobacteria or Salmonella [5-8]. In this regard, integrated miRNA- as well as mRNA-transcriptome analysis by means of microarrays and reverse transcription quantitative PCR (RT-qPCR) allowed us to show that in mycobacterial infections of human macrophages caspases 3 and 7 are under the con- trol of let-7e and miR-29a, respectively [8]. Many methods such as microarrays, RNAseq or RT- qPCR are used to study mRNA as well as miRNA expres- sion of cells or tissues under a given condition. Quantifi- cation of miRNAs but also other small non-coding RNAs by means of RT-qPCR was established during the last decade based on several detection strategies. In this regard, we developed a protocol called miR-Q, which relies on reverse transcription (RT) of miRNAs using spe- cific oligonucleotides. The generated cDNA is quantified by means of a novel qPCR protocol using three oligonu- cleotides based on SYBR Green detection chemistry [9]. The protocol can easily be customised to detect and quantify any class of non-coding small RNAs. Besides the frequent application of miR-Q in our lab, the protocol was adopted by others for quantification of small RNAs e.g. in immunity-, virus-, metabolism- or cancer-related RNAi research [10-13]. The miR-Q protocol enables highly specific quantification of single miRNAs providing a high sensitivity. Since RT-qPCR remains the gold-stan- dard for accurate quantification of gene expression, such arrays are increasingly employed for mid-throughput quantification of gene expression providing high sensitiv- ity coupled with accuracy. While arrayed quantification of mRNAs is performed easily, the short length of miR- NAs and underlying detection chemistries require well- conceived strategies. A few commercial miRNA RT- qPCR arrays are currently available, which are rather costly. Based on the stem-looped qPCR approach of Applied Biosystems Tang and colleagues reported the 220-plex miRNA expression of a single cell [14]. Another group reported a multiplex quantification of miRNAs involving the purification of multiplex PCR amplicons followed by uniplex analysis on microfluidic chips [15]. However, until today there are no non-commercial and custom protocols for miRNA RT-qPCR arrays. This prompted us to advance the existing miR-Q protocol allowing more versatile and fully automatable arrayed quantification of any class of eukaryotic small non-coding RNAs. Compliant with the MIQE guidelines and nomen- clature [16], we provide here a reliable and cost-effective method enabling arrayed quantification combined with accurate normalisation of miRNA expression. Page 2 of 11 Results and discussion Multiplexed reverse transcription of miRNAs enables miR- Q arrays After the miR-Q approach was established and published, several labs worldwide approved its performance for their research by adopting the protocol. Over the course of the last three years we further established miR-Q assays for several miRNAs according to different research projects [8,17,18]. Another 21 newly established assays are pre- sented here for the first time including a set of five snRNA specific assays serving as reference genes. Taken together, we have developed 44 small RNA specific miR-Q assays and the particular oligonucleotide sequences are presented in table 1. As reported earlier [9], we usually determine the dynamic range of miR-Qs by spiking bacterial total RNA with serial dilutions of synthetic miRNA in single assays (2 nM-2 fM final concentration regarding the RT- qPCR). Most miR-Q assays possess a dynamic range of 5 to 6 orders of magnitude. According to our experience the lower spectrum of calibration curves however corresponds to the naturally occurring and physiological miRNA con- centration of a cell. Therefore, we determined the Y-inter- cept, slope and r2 for all presented miRNA specific assays ranging from 20 pM to 20 nM (Additional file 1). The dynamic range of reference snRNA specific assays was determined in a range between 100 and 0.01 ng cDNA. As it is shown in Figure 1, all assays possessed proper efficien- cies calculated from the slopes of calibration curves. Figure 1 moreover shows that there was no statistically significant difference between the efficiencies of miRNA- and refer- ence snRNA-specific miR-Q assays proven by a paired t test (P = 0.6836). According to our experience, methods such as microarrays or next-generation-sequencing can be applied for the gen- eration of snap shots of a cell or tissue miRNAome [8,19]. However, we were interested in a fast, cost-effective, feasi- ble and accurate alternative. Accordingly, we advanced the conventional miR-Q protocol and reaction conditions allowing increased performance of miRNA RT-qPCR arrays (miR-Q arrays). For this purpose, we evaluated mul- tiplexed RT reactions with different numbers of specific RT oligonucleotides per reaction (RT6-’miRNA’) and vary- ing respective final concentrations. In contrast to the con- ventional miR-Q RT, the reaction time was extended and a higher amount of reverse transcriptase was used for the miR-Q arrays. A 31-plex RT turned out to be most suita- ble because it allowed detection of the respective number of small RNAs and negative controls (NC) measured in triplicate when using a 96-well format. A miR-Q array lay- out is exemplified in Figure 2A. This layout allowed the detection of 26 different miRNAs within the same PCR run and simultaneously quantifying the expression of 5 additional reference snRNAs (SNORD44, 47, 48, 52 and