Mir xtm mirna first strand synthesis

Total RNA was extracted from tissues and cells using TRIzol reagent (Takara, Dalian, China) based on the manufacturer’s instructions. The complementary DNAs (cDNAs) corresponding to the lncRNAs and mRNAs of interest were reverse-transcribed from 1 μg of total RNA using PrimeScript RT-polymerase (Takara). The cDNAs for the miRNAs of interest were synthesized using Mir-X^Tm miRNA First-Strand Synthesis (Clontech, Dalian, China). The qRT-PCR was performed using SYBR-Green Premix (Takara) with specific PCR primers (Sangon Biotech Co., Ltd, Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and RNU6 (U6) were used as internal controls due to their stability across all groups studied. Fold changes were calculated by means of the 2^−ΔΔCt method. Primer sequences are listed in Supplementary Table S3.

1μg of RNA isolated from peripheral blood was reverse transcribed and used for miRNA quantification using Mir-X^TM miRNA First-Strand Synthesis and TB Green qRT-PCR kit (Takara Bio Inc.). U6 was used as an internal control. For mRNA quantification, about 1μg of RNA isolated from peripheral blood was converted to cDNA with the cDNA Reverse Transcription Kit (Applied Biosystems, USA). Quantitative RT-PCR (qRT-PCR) was carried out on RealPlex PCR system (Eppendorf, USA) with SYBR Premix Ex Taq^TM (TaKaRa Bio Inc.). GAPDH was used as the internal control. Statistical analysis was performed using GraphPad 7.0. The primers used are enlisted in Additional file 1.

Total pulp RNA was extracted using the RNAplant Plus Reagent kit (Tiangen, Beijing, China) based on the manufacturer’s protocol. Three biological replicates of each sample were kept at -80 °C and used for the extraction of RNA. The purity and integrity of the total RNA were determined by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Madison, WI, USA) and gel electrophoresis using the PrimeScriptTM RT Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions; 2.0 µg of each RNA sample was used to generate first-strand cDNA for gene isolation. For miRNA reverse transcription, Mir-XTM miRNA first-strand synthesis and the TB Green Real-Time PCR (qPCR) kit were used (TaKaRa, China). The QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Singapore) was applied to perform quantitative reverse transcription PCR (qRT-PCR). The qRT-PCR patterns were performed at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 20 s. As an internal control U6 gene, DkActin (accession no. AB473616) was used. The forward miRNA primer was designed using the fasta sequence, and universal primer was used as a reverse primer (Table S1).

Cell fractionation assay in accordance with PARIS^TM kit (Cat number: AM 1921) as followed: First, Cells washed twice with ice-cold PBS were resuspended with 300 μl cold Cell Fractionation Buffer (10 U/ml RNase inhibitor) on ice for five minutes and then were centrifuged at 500 g in 4 °C centrifuge for 5 min. Then the supernatant of the extract was shifted into a new microcentrifuge tube and centrifuged again at 500 g centrifuge for one minute in 4 °C. The cytoplasmic fraction (the supernatant) was shifted again into a new tube. Then, the nuclear fraction was washed once in cold cell fractionation buffer and resuspended and centrifuged 500 × g at 4 °C for one minute. Remove and discard the supernatant. The nuclear extract was lysed with 300 μl cell disruption buffer and RNA was extracted from the nuclear pellet in accordance with the manufacturer’s protocol.
For assessing the nuclear and cytoplasm HIFAL abundance, we added the synthetic cel-mir-39 (at a final concentration of 25fmol)^{52 (link)} as exogenous internal reference in the nuclear and cytoplasm lysate which was mixed with the equal volume of 2X Lysis/Binding Solution. QRT-PCR was performed using Mir-x^TM miRNA First-Strand Synthesis and TB Green^TM qRT-PCR(Takara).

Using Trizol (Invitrogen, USA) for RNA extraction from the RCC cell lines according to the manufacturer's instructions. miRNA was reverse-transcribed into cDNA using MiR-XTM miRNA first-strand synthesis (Takara, JPN). The total RNA was reversed transcribed into cDNA using PrimeScript RT Master Mix (Takara, JPN). A standard SYBR Green PCR kit (Takara, JPN) was used to perform qRT-PCR. The following forward and reverse primer sequences are shown in Table S1.

Total RNA were extracted from HCC cells or frozen liver tissues using RNAiso Plus (Takara, Japan). The reverse transcription (RT) and qPCR were performed with Mir-X^TM miRNA First-Strand Synthesis, PrimeScript RT Master Mix and SYBR Premix Ex Taq II (Takara) according to the manufacturer’s instruction. Each experiment was performed in triplicate. RT-qPCR of miR-200c detection was done as previously described^{40 (link),48 (link),49 (link)}. U6 and GAPDH were used as housekeeping genes normalization for miR-200c and other transcripts, respectively, and the relative expression level was calculated with 2^^−ΔΔCt methods. The primers were list in the Supplementary table S3.