Miscript 2 rt kit

For mRNA analysis, reverse transcription was performed with 0.1–1 µg RNA using Qiagen^® Quantitect RT Kit following manufacturer’s instructions to create cDNA. For miRNA analysis, reverse transcription was performed with 0.1–1 µg RNA using miSCRIPT II RT Kit, HiSpec buffer, (Qiagen) following manufacturer’s instructions to create cDNA. Generated cDNA was used for quantitative real-time PCR analysis using Power SYBR^® Green PCR Master Mix (Applied Biosystems) and quantified on the 7900HT (Applied Biosystems) or QuantStudio5 (Applied Biosystems). See Table S1 for mRNA primer sequences. Where appropriate, relative mRNA expression was determined via normalisation to the housekeeping gene Hprt and the relevant control group (see Figure legends). All miRNA and snoRNA primers were purchased from Qiagen for use with the miSCRIPT II RT Kit. Where appropriate, relative miRNA expression was determined via normalisation to the housekeeping snoRNA RNU6B and the relevant control group (see Figure legends).

TRIzol Reagent (Invitrogen, USA) can be used for total RNA extraction from tissues and cells. Total RNA in serum and exosomes was extracted with QIAzol Lysis Reagent (Invitrogen, USA). The miScript II RT Kit (Qiagen, Germany) can be used to reverse transcribe mRNAs, and the miScript SYBR Green PCR Kit (Qiagen, Germany) can be used to detect them. Similarity, the miScript II RT Kit (Qiagen, Germany) is used for miRNA reverse transcription, and the SYBR Green PCR Kit (Qiagen, Germany) can be used to detect reverse transcriptional mRNA. Quantitative analyses can use CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were repeated three times. The internal reference of mRNAs is GAPDH. The internal parameter of miRNAs is U6. Sangon (Shanghai, China) (Supplementary Table S2) designed and synthesized primers for mRNAs. The primers for miRNA detection were purchased from Qiagen.

At 96 h post electroporation, RNA was isolated from worms using RNAiso plus (Takara) according to the manufacturer’s protocol except for an extended overnight precipitation in isopropanol at -80°C. First strand cDNA was produced from 100 ng RNA using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara) with either a miRNA specific primer (2 μM, 0.5 μL) or random primer combined with Oligo d(T) as described above. qPCR was then carried out to determine the expression of miR-750 targets using the primers described in S9 Table. S. japonicum NADH was used as an internal control. Part of the total RNAs were reversely transcribed using a miScript II RT Kit (Qiagen) to determine the expression of miR-750 using an miScript II RT Kit (Qiagen) as described above. All reactions were performed in at least triplicate. The 2^-ΔCt method was used to calculate the relative expression levels.

Based on the changes of gene expression found in the Neurological qPCR arrays, a gene of interest, miR-652, was investigated based on its association with autism spectrum disorder. A more nuanced measurement of this gene’s expression was performed using each biological replicate. Isolated RNA from each mouse replicate was reverse transcribed to form miRNA-enriched cDNA using Qiagen miScript II RT Kit and the manufacturer’s instructions.
The expression of mmu-mir-652-3p was measured using Qiagen 10x miScript Mm_miR-652_3 (MS00012425) primer assay. qPCR was completed using QuantiTect SYBR Green PCR Master Mix according to the vendor’s instructions. Samples were compared to a control primer assay (RNU6).
In order to measure expression of an mRNA target of mmu-mir-652-3p, isolated RNA from each mouse brain replicate was reverse transcribed to form mRNA-enriched cDNA using Qiagen miScript II RT Kit and the manufacturer’s instructions. The mRNA-enriched cDNA was measured by qPCR using QuantiTect SYBR Green PCR Master Mix. No mice were excluded from these experiments. There were four biological replicates and four technical replicates for each reaction.

To quantify miR-449b, total miRNA was purified using the mirVanaTM miRNA Isolution Kit (Qiagen, Gaithersburg, MD, USA), and reverse transcription was performed using the miScript II RT Kit (Qiagen). The expression of miRNA-449b was determined using the miScript SYBR Green PCR Kit (Qiagen). Primer sequence upstream of miR-449b: GGGAGGCAGTGTATTGTTAGCTG. Primer sequence upstream of U6: CGCTTCGGCAGCACATATACTA. Downstream primers were obtained using the miScript II RT Kit (Qiagen).