Miscript reverse transcriptase mix

Reverse transcription (RT) reactions were performed on all obtained extractions using the miScript II RT kit (Qiagen, Hilden, Germany) in a 5 μl RT reaction: 1 μl 5× miScript HiSpec Buffer, 0.5 μl 10× miScript Nucleic Mix, 0.5 μl miScript Reverse Transcriptase Mix, and 0.5 μl of 0.1 pM synthetic cel-miR-39 (Qiagen, Hilden, Germany), to control for efficiency of RT and polymerase chain reactions (PCRs), as well as to monitor the presence of potential endogenous inhibitors derived from plasma samples. These components were combined and composed the master mix. After mixing by inversion and centrifugation, the master mix was aliquoted into 0.2 ml RNase-free strip-tubes, followed by the addition of 2.5 μl input RNAs.
In module 1 and module 3, RT reactions were carried out on the Biometra thermal cycler (M-Medical) using the following conditions: 37 °C for 60 min, 95 °C for 5 min, and then held at 4 °C. cDNAs were then diluted 1:10 using nuclease-free water. Diluted RT products were stored at −20 °C prior to real-time PCR. In module 2 (Sec. III B), RT reactions were carried out on the S1000™ thermal cycler (Bio-Rad) using the conditions: 37 °C for 60 min, 95 °C for 5 min, and held at 4 °C. cDNAs were then diluted 1:5 using nuclease-free water (Qiagen).

The qRT-PCR was performed as described by He et al. [61 (link)]. For miRNA, reverse transcription was performed with 1 μg RNA using the miScript reverse transcriptase mix (Qiagen, Beijing, China) according to the manufacturer’s protocol. The miRNA specific forward primer is the same as the mature miRNA sequence. U6 was used as an internal control. Real-time PCRs of miRNAs were performed using the miScript SYBR Green PCR kit (Qiagen, Beijing, China) with the specific primers and the universal primer provided in the kit following the production instructions. All primer sequences are listed in Table S25.

Anaesthetic ether, xylene, chloroform and formalin were purchased from Beijing Chemical Works (Beijing, China). Ethanol was obtained from Shandong LIRCON Medical Technology Co., Ltd. TRIzol RNA isolation reagent was purchased from Invitrogen (Carlsbad, USA). miScript HiSpec Buffer, miScript Nucleics Acid Mix, miScript Reverse Transcriptase Mix, RNase-free water oligo primer, reaction buffer, dNTPs, revertase, and RNase inhibitor were obtained from Qiagen (Dusseldorf, Germany).

Reverse transcription was carried out using a miScript II RT Kit (Qiagen). A 20 μL reverse transcription reaction was prepared with 2.2 μL of eluted miRNA, 4 μL 5× miScript HiSpec Buffer (Qiagen), 2 μL 10× miScript Nucleics Mix (Qiagen), 9.8 μL RNase-free water, and 2 μL miScript Reverse Transcriptase Mix (Qiagen). The reaction was incubated at 16 °C for 60 min followed by 95 °C for 5 min. The reverse transcription reaction was then diluted with 200 μL RNase-free water. Triplicates of qPCR reactions were carried out using miScript SYBR Green PCR Kit (Qiagen) and run on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). The reaction contained 2 μL diluted cDNA, 12.5 μL 2× QuantiTect^® SYBR Green PCR Master Mix (Qiagen), 2.5 μL 10× miScript Universal Primer (Qiagen), 10× miScript Primer Assay (Qiagen) for the target miRNA, and 5.5 μL RNase-free water in a final volume of 25 μL. The reaction mixtures were incubated for 15 min at 95 °C, followed by 45 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. Standard curves were generated from a series of dilutions for the target miRNA (synthetic miScript miRNA Mimics from Qiagen) for each plate (Supplementary Fig. 13). Quantitation cycle (Cq) values were acquired and analyzed using StepOne™ Software v2.3 in accordance with the MIQE guidelines^{43 (link)}.

The expression profiles of six randomly-selected miRNAs were investigated with qPCR to validate their expression changes. Total RNA (500 ng) was converted to cDNA using miScript reverse transcriptase mix (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. qPCR was carried out using an Applied Biosystems 7300 Real-Time PCR System according to the standard protocol. CDNA samples were diluted to 1:150; 5 μL were used for each real-time PCR reaction. The 20-μL PCR mixture included 10 μL SYBR Premix Taq (2×), 0.4 μL miRNA-specific forward primers (10 μM), 0.4 μL miScript universal primer (10 μM), and 1 μL PCR template (cDNA). The PCR thermal program was 50 °C for 2 min, followed by 40 cycles of 95 °C for 2 min, 95 °C for 15 s, and 60 °C for 30 s. Melting curve analysis was performed after amplification. Standard curves for endogenous control and for all miRNAs were constructed using serial dilutions of a pooled cDNA sample. Standard curves were used to determine the quantity of the selected miRNAs and reference genes. Relative miRNA expression levels were calculated using the 2^−ΔΔCt method. Each sample was run in triplicate. SnRNA U6 was used as an endogenous control for qPCR of miRNAs.

Each RNA sample was checked for genomic DNA contaminations that could obscure the measurement. One microgram of total RNA was converted to cDNA using the miScript Reverse Transcriptase mix (Qiagen) following the manufacturer’s instructions. Quantitative RT‐PCR was performed in triplicate with a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) using the miScript SYBR Green PCR Kit according to the manufacturer’s protocol. Molecules of the QuantiTect Primer Assays (Qiagen) were used as primers: hsa‐miR‐1225‐3p, hsa‐miR‐1260_1, hsa‐miR‐4516_1 and hsa‐RNU6‐2_11, the last used for normalisation purposes as suggested by Qiagen. Data were analysed using the lightcycler software (Roche Diagnostics).