miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample.
BACKGROUND
MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.


RESULTS
Here, we present the miR-Q assay as a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as miRNA molecules. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM miRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays. Using this approach, we quantified the expression of let-7b in different human cell lines as well as miR-145 and miR-21 expression in porcine intestinal samples.


CONCLUSIONS
miR-Q is a cost-effective and highly specific approach, which neither requires the use of fluorochromic probes, nor Locked Nucleic Acid (LNA)-modified oligonucleotides. Moreover, it provides a remarkable increase in specificity and simplified detection of small RNAs.
Sharbati-Tehrani
Soroush
S
Institute of Veterinary-Biochemistry, Freie Universität Berlin, Oertzenweg 19 b, 14163 Berlin, Germany. sharbati@zedat.fu-berlin.de
Kutz-Lohroff
Barbara
B
Bergbauer
Ramona
R
Scholven
Jutta
J
Einspanier
Ralf
R
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013552:Swine / Q000235:genetics
D035683:MicroRNAs; C487931:mirnlet7 microRNA, human