Technique / Molecular Biology / RNA transcriptional post-transcriptional regulation / Northern blot hybridization

MicroRNAs (miRNAs) are small, endogenous, non-coding RNA species, about 21 nucleotides in length, which modulate the expression of animal and plant target genes at the post-transcriptional level. It has been shown that miRNA based gene regulation plays a pivotal role in pathways involved in growth and development. Understanding miRNA mediated processes requires new technologies enabling efficient detection of small RNA species. Here we report the optimization of a miRNA Northern blot detection method based on LNA (locked nucleic acid)-modified oligonucleotide hybridization. This technique allows sensitive and highly specific detection of mature miRNAs.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

OBJECTIVE: To determine relative amounts of mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3 in articular cartilage and synovial membrane samples from healthy equine joints and joints with osteoarthritis (OA) and to compare results of Northern blot hybridization with results of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. SAMPLE POPULATION: Articular cartilage samples from 8 pairs of joints (1 with OA and 1 healthy) from 6 horses and synovial membrane samples from 6 pairs of joints from 5 horses. PROCEDURE: RNA was extracted from samples by use of a modified Trizol procedure. Northern blot hybridization and the RT-PCR assay were performed; results were quantitated by use of glyceraldehyde 3-phosphate dehydrogenase as an internal standard. RESULTS: Articular cartilage samples from joints with mild or moderate OA yielded less total RNA than samples from joints with severe OA. Northern blot hybridization indicated that type-II collagen mRNA expression in articular cartilage samples from joints with OA was significantly greater than expression in samples from healthy joints. The RT-PCR assay identified low levels of MMP3 mRNA expression in 4 of 8 sets of articular cartilage samples and 4 of 6 sets of synovial membrane samples, whereas Northern blot hybridization identified MMP3 mRNA expression in only 1 of 6 sets of articular cartilage samples and 1 of 6 sets of synovial membrane samples. CONCLUSIONS: A RT-PCR assay is more sensitive than Northern blot hybridization for detection of MMP3 mRNA expression in articular cartilage and synovial membrane and requires smaller samples.