Miscript rt kit

For circ_0066147 and E2F2 detection, cDNA was obtained according to the standard protocols using a SuperScript VILO cDNA Synthesis kit (Invitrogen) with random primers (Invitrogen); qRT-PCR analysis with SYBR Green (Qiagen) and designed primers (Table 1) was run in triplicate on a DNA Engine Opticon Monitor System (Bio-Rad, Glattbrugg, Switzerland). For miR-326 quantification, the miScript RT kit (Qiagen), miScript SYBR Green PCR kit (Qiagen) and designed primer (Table A1) were used. Data were analyzed by the comparative Ct (2^−ΔΔCt) method [23 (link)] and presented as a corrected value by normalizing with the expression of the reference gene β-actin or U6.

Total RNAs and miRNAs were extracted from sera samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, and RNA concentration and purity were evaluated spectrophotometrically at 260 and 280 nm. RNA integrity was visually confirmed by agarose gel electrophoresis. Synthesis of cDNA was completed by reverse transcription reaction using a miScript RT Kit (Qiagen, Hilden, Germany). In the second step, cDNA was amplified for miRNA expression using a miScript Primer Assay for miRNA amplification (Rn_miR-137_1 and Rn_miR-106b-5p_1 miScript Primer Assay). The At_U19_1 and Rn_ GAPDH genes were used as reference housekeeper genes. The thermal cycling was adjusted according to the manufacturer’s instructions. The PCR analysis was conducted on a Rotor-Gene Q 5plex high resolution melting (HRM) platform (Qiagen, Hilden, Germany). The fluorescence data were collected at the extension step. Following amplification, gene expression was calculated using the 2^∆∆Ct method.

Small RNA-rich samples were isolated from cells with the TRIzol reagent or mirVana miRNA Isolation kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. cDNAs were synthesized with a miScript RT kit (Qiagen GmbH, Hilden, Germany), and PCR was performed with a LightCycler using miScript SYBR green PCR kit (Qiagen GmbH) as described previously [35 (link), 55 (link)]. Forward sequences for the miRNAs primer are described in Table 1.

Total RNA was isolated from primary T cells or Jurkat cells using the RNeasy Mini Kit (Qiagen). 100 ng-200 ng reverse transcription reactions for cDNA synthesis were carried out using the miScript RT Kit (Qiagen) according to the protocol for subsequent quantification of miRNAs and mRNAs.
Real-time PCR (RT-PCR) analysis of gene (mRNA) and miRNA expression was carried out using Quantitect SYBR Green Primer Assays (Qiagen, for mRNAs) and miScript SYBR Green Primer Assays (Qiagen, for miRNAs) and the Bio- Rad CFX96 real-time cycler. For some gene expression analysis, we used the RT² qPCR Primer Assays (Qiagen). βeta-2 microglobulin (β2M) was used as an internal control for gene expression. The small nuclear RNA, U6, was used as an internal control for miRNA expression. For extravesicular miRNA expression, C. elegans miR-39 which detects the Spike-In Control added to samples during EV RNA isolation was used for normalization of RT-PCR results for miRNA expression. For data analysis, we used the ΔΔC_T method of relative quantification.

For miRNA analyses, total extracted RNA was retrotranscribed using the miScript RT kit (Qiagen, Hilden, Germany), and 100 ng of obtained cDNA were analyzed by the “Human Inflammatory Response & Autoimmunity” microarray (Qiagen, Hilden, Germany), able to detect and quantify 84 different miRNAs simultaneously. In addition, 29 further individual assays were performed, including miRNAs specifically involved in pregnancy and not included in the array: miR18, miR21-5p, miR22, miR24, miR30b-5p, miR31, miR92, miR125-3p, miR125b-5p, miR145-5p, miR155_1, miR155-2, miR196b-5p, miR199b-5p, miR200_1, miR200_2, miR222, miR374a-5p, miR378a-3p, miR422, miR423-5p, miR424-5p, miR449a, miR517, miR572, miR575, miR1207-5p, miR3663-3p, miR4306 and miR5739. miRTC_1, and SNORD11 were used as controls (Qiagen, Hilden, Germany). Amplification results were analyzed and normalized by a specific Qiagen software, to obtain comparable values between control and infected cells at each time post infection.

Total RNA was isolated from thymic lymphoma tissues and normal thymus tissues using Trizol (Invitrogen, USA) according to the manufacturer’s instructions (14 (link)). Reverse Transcription and qPCR was subsequently performed in triplicate using the mi Script RT Kit and mi Script PCR system (Qiagen). Relative quantities of each miRNA were calculated using the ΔΔCt method after normalization with endogenous reference U6-small nuclear RNA.
Normal and RITL tissues were sent to Capital Bío Company (Beijing, China) for miRNA biochip analysis. The miRNA gene chip used in this experiment is a commercial miRNA oligonucleotide expression profile designed by Agilent based on the miRNA sequence of the database (Sanger miRBase release 16.0: http://microrna.sanger.ac.uk/sequences/, AgilentTechnolo gies) Chip (8×60K). The chip contains probes for human miRNA (1,205 in total) and human virus miRNA (142 in total), which basically covers the currently known human miRNA. This chip is highly sensitive and specific, and can also distinguish mature and precursor miRNAs.
The mRNA chip covers 30,656 human genes and transcripts. It basically covers mRNAs with known functions. The design of each probe is optimized through trial and error, and the average data has more reliable statistical significance, which improves the accuracy of chip detection.