Tianscript m ml 5

For measurement of mRNA levels, total RNA from tissue samples was isolated using RNAprep Pure Tissue kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Random-primed cDNAs were generated by reverse transcription of total RNA with TIANScript M-MLV (Tiangen). A real-time PCR analysis was performed with the ABI Prism 7000 Sequence Detection system (Applied Biosystems) using SYBR® Premix-Ex TagTM (Takara, Dalian, Chnia). GAPDH was used for an internal control. Primer sequences for these mRNAs were listed in Table S2.
Mature miRNAs from tissue samples, serum and exosomes were extracted using miRcute miRNA Isolation kit (Tiangen). Pre-miRNAs from tissue samples were extracted using RNAprep Pure Tissue kit (Tiangen). poly(A) modification and first-strand cDNA synthesis were performed with miRcute miRNA first-strand cDNA synthesis kit (Tiangen). qRT-PCR analysis for mature miRNA was conducted using miRcute miRNA qRCR detection kit (Tiangen). For measurement of miRNA or pre-miRNA levels in the tissue samples,18S rRNA was used as an internal control. The miRNA levels in serum and exosomes were determined using absolute quantitative real-time RT-PCR described previously^{40 (link)}. Primer sequences for these miRNAs were listed in Table S2.

TRIzol solution (Invitrogen) was used to extract or isolate total RNA form glioma tissues or cells. Reverse transcription was finished by using PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). To detect the expression level of miRNA, the reverse transcription of miRNAs was carried out with TIANScript M‐MLV (Tiangen, Beijing, China). qRT‐PCR was conducted on a LightCycler 480 instrument (Roche, Basel, Switzerland) using SYBR Premix Ex Taq II (TaKaRa). The conditions for thermal cycle were shown as follows: 95°C for 30 seconds followed by 40 cycles at 95°C for 5 seconds and at 60°C for 30 seconds. The specific primers of genes were bought from Invitrogen. GAPDH and U6 were separately used as the internal control. The relative expression levels of genes were calculated by using the 2^−ΔΔCt method.