Purelink rna micro to midi kit

Total RNA from female inflorescences (FI) and top internodes of the stem (TI) at 60 DAS were extracted using the PureLink RNA-micro-to-midi Kit (Invitrogen Corp., Carlsbad, CA, USA), as described by the manufacturer. The purity and integrity of the RNA samples were evaluated by electrophoresis on 2% RNase-free agarose gels, spectrophotometry (A260/A280 and A260/A230 ratios) using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and quality analysis using the Agilent 2100 Bioanalyzer system (Agilent technologies, Inc, Santa Clara, CA, USA).

Mel-Juso cells transfected with miRVec-125b or the miRVec-control were collected and washed twice in PBS. Murine tumour tissue samples were homogenized using a TissueLyzer II (Qiagen, Valencia, CA, USA) and processed as previously described [40 (link)]. Small RNAs were purified using the mirVana Isolation Kit (Ambion, Foster City, CA) and the PureLink RNA Micro to Midi Kit (Invitrogen) according to the manufacturer’s instructions. The concentration of RNA was measured spectrophotometrically using NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE), and RNA integrity was confirmed with Agilent 2100 Bioanalyzer using Agilent Nano RNA kit (Agilent Technologies, Santa Clara, CA). miR-125b expression was measured in the samples containing the same amount of RNA with quantitative qRT-PCR assay (TaqMan TM microRNA Reverse Transcription kit-4366596, and TaqMan Universal PCR Master mix-4324018, Applied Biosystems, Foster City, CA) and validated primer sets (Applied Biosystems) according to the manufacturer’s instructions. MiR-191 was used as a reference for the normalization of qRT-PCR-data. The q-PCR was performed in triplicates using a 7900HT Fast Real-Time PCR System (Applied Biosystems).