For RNA isolation from cells or rodent tissues, the miRNeasy Mini Kit (Qiagen) was used. First-strand cDNA was synthesized using SuperScript IV (Life Technologies) according to the manufacturer’s protocol. For qRT-PCR analyses, the LightCycler 480 SYBR Green I Master kit (Roche) was used on a LightCycler 480 Instrument II (Roche). All amounts were normalized to their GAPDH controls. miRNA expression level was assessed using the miQPCR method as previously described (19). For human samples, midbrain blocks were transferred shortly to a cryostat for the sake of having controlled temperature conditions during sampling (−20°C). Tissue punches were extracted from the frozen blocks using a 20-G Quincke Spinal Needle (Becton Dickinson), and the sampled material (around 20 mg) was stored into ribonuclease (RNAse)/deoxyribonuclease-free tubes. Total RNA was isolated from the samples using TRI Reagent (Sigma-Aldrich) following the manufacturer’s protocol. RNA precipitates were reconstituted in 15 μl of nuclease-free water, and the samples were cleaned and concentrated using the column-based RNA Clean & Concentrator-5 KIT (Zymo Research). RNA (1 μg) from each patient sample was reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). cDNA samples were diluted 1:3 in nuclease-free water and kept at −20°C until further use. For human GAPDH, we used the primers “Hs_GAPDH_1_SG” provided in the QuantiTect Primer Assay (QT00079247). Unless otherwise stated, all other primers used in this study are summarized in data S1J.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.