Nanophotometer spectrometer

Total RNA was extracted from tissues or PC cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The quantity and quality of the RNA were determined using a NanoPhotometer spectrometer (ImplenInc, CA, USA). MicroRNA expression profiles of PC specimens and adjacent noncancerous tissues (n = 5 per group) were analyzed using the Affymetrix Genechip miRNA 4.0 array (KangChen Biotech, Shanghai, China) according to the manufacturer’s instructions. Data were obtained and analyzed with AGCC software (Affymetrix, Santa Clara, CA, USA). Fold change greater or equal to 2 have been considered as significant change.
Total RNA (500 ng) was reverse transcribed to get the first-strand cDNA using the Prime Script RT Reagent Kit (Takara, Dalian, China). Quantitative PCR assays were carried out on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq (Takara, Dalian, China). Results were normalized to the expression of GAPDH. The mirVanaTM qRT-PCR microRNA Detection Kit (Ambion Inc., Austin, TX, USA) was used to assess the levels of miR-3064 according to the manufacturer’s instructions. U6 was used for data normalization.

RNA extraction and sequencing of 96 brain samples were performed by Novogene Beijing. RNA degradation and contamination were monitored using 1% agarose gels, and RNA purity was assessed using a NanoPhotometer spectrometer (IMPLEN, CA, USA). RNA concentration and integrity were measured using a Qubit 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA) and an Agilent Bioanalyzer 2100, respectively. Sequencing libraries preparation used 3 μg RNA per sample and cluster formation was conducted following manufacturer’s recommendations (TruSeq PE Cluster Kit v3-cBot-HS, Illumina). Sequencing was performed on an Illumina HiSeq platform. The outcome generated paired-end reads with a fragment length of 300–500 bp and an average paired end length of 150 bp, for a total of 519 millions of raw paired-end reads sequenced (~ 5.4 million paired-end reads per sample).

Each stage had three replicates for RNA extraction, and each replicate contained approximately 500 mg of larvae. We carried out RNA extraction following the instructions of the TRIzol Kit (Invitrogen, CA). RNA quality was examined by electrophoresis through 1.2% agarose gels to check for possible degradation and contamination. RNA purity was monitored on a NanoPhotometer spectrometer (IMPLEN, CA) using default parameters. Integrity and concentration and of RNA was measured using a Qubit RNA Assay Kit in a Qubit 2.0 Flurometer (Life Technologies, CA), and the RNA 6000 Nano Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA), respectively. Fifteen RNA samples (five stages × three replicates) that satisfied all requirements of the quality test were selected for library preparation.
RNA (3 μg per sample) was used as the input material for the RNA sample preparations. Sequencing libraries were constructed according to the manufacturer’s instructions using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB). After clustering of the index-coded samples, we performed RNA sequencing of each library on an Illumina HiSequation 2500 platform to generate 100-bp single-end (SE) reads. This procedure was carried out by Novogene Bioinformatics Technology Co. Ltd (Tianjing, China).