OBJECTIVE: To optimize and evaluate the modified RNA-tailing and primer-extension RT-PCR method in relative quantification of microRNAs (miRNAs) in several kinds of tissues. METHODS: Small-sized RNAs (<200 bp) were extracted and polyadenylated by poly(A) polymerase. One-base anchored oligo-dT primers with 40 nt extension at their 5'-ends were used to reversely transcribe the poly(A)-tailed miRNAs. miRNAs were then amplified by SYBR Green real-time PCR using miRNA specific primers. RESULTS: This method had a high dynamic range and could detect low abundant miRNAs. The assay was capable of discriminating between related miRNA family members that differed by subtle sequence differences at 3' ends, but it could not distinguish those differed in the middle of their sequences. Fifteen mature miRNAs were differentially expressed in rat heart, liver, and cortex,which were in accordance with published results by other methods. The miRNA expression signature in hippocampus was different from that in cortex. miR-122a expression was higher and miR-124a expression was lower in hippocampus than in cortex. A single dissociation peak on the thermal melting curve and a single DNA band about 80 bp on 3.5% NuSeive agarose gel signified the amplification specificity for these miRNAs. CONCLUSION: This method enables fast and sensitive relative quantification for most miRNAs and can identify potential biomarkers specific to tissues or diseases.
OBJECTIVE: To optimize and evaluate the modified RNA-tailing and primer-extension RT-PCR method in relative quantification of microRNAs (miRNAs) in several kinds of tissues. METHODS: Small-sized RNAs (<200 bp) were extracted and polyadenylated by poly(A) polymerase. One-base anchored oligo-dT primers with 40 nt extension at their 5'-ends were used to reversely transcribe the poly(A)-tailed miRNAs. miRNAs were then amplified by SYBR Green real-time PCR using miRNA specific primers. RESULTS: This method had a high dynamic range and could detect low abundant miRNAs. The assay was capable of discriminating between related miRNA family members that differed by subtle sequence differences at 3' ends, but it could not distinguish those differed in the middle of their sequences. Fifteen mature miRNAs were differentially expressed in rat heart, liver, and cortex,which were in accordance with published results by other methods. The miRNA expression signature in hippocampus was different from that in cortex. miR-122a expression was higher and miR-124a expression was lower in hippocampus than in cortex. A single dissociation peak on the thermal melting curve and a single DNA band about 80 bp on 3.5% NuSeive agarose gel signified the amplification specificity for these miRNAs. CONCLUSION: This method enables fast and sensitive relative quantification for most miRNAs and can identify potential biomarkers specific to tissues or diseases.