Taqman microrna assay protocol

miRNA was extracted with miRNeasy system for purifying total RNA (Qiagen). Expression analysis was performed by using TaqMan probes (Applied Biosystems; Foster City, CA) for real-time PCR. cDNA was synthesized from total RNA using gene-specific primers according to the TaqMan MicroRNA Assay protocol (Applied Biosystems; Foster City, CA). Reverse transcriptase reactions contained 10 ng of RNA samples, 50 nM stem-loop RT primer, 1× RT buffer, 0.25 mM each of dNTPs, 3.33 U/µl MultiScribe reverse transcriptase and 0.25 U/µl RNase inhibitor (cDNA Archive kit from Applied Biosystems). The 7.5 µl reactions were incubated for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and then held at 4°C. Real-time PCR was performed using an Applied Biosystems 7300 Sequence Detection system. The 20 µl PCR included 0.67 µl RT product, 1× TaqMan Universal PCR master mix and 1 µl of primers and probe mix of the TaqMan MicroRNA Assay protocol (Applied Biosystems; Foster City, CA). The reactions were incubated in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60° for 10 min. The CT values were determined using default threshold settings. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.

miRNA was quantified using reverse transcription and quantitative real-time PCR (qRT-PCR). Small RNAs from cell and tissue samples were prepared using a TRIzol and miRNeasy mini kit (Qiagen, Valencia, USA) according to the manufacturer's instructions. cDNA was synthesized from total RNA using gene-specific primers according to the TaqMan MicroRNA Assay protocol provided by the manufacturer (Applied Biosystems). Quantitative PCR for miR-1/133a/206 was performed using an Applied Biosystems 7300 Sequence Detection system. The 10 μL PCR reaction contained 0.67 μL of reverse transcription product, 1 x TaqMan Universal PCR master mix, and 1 μL of primer and probe mix, according to the TaqMan MicroRNA Assay protocol (Applied Biosystems). Samples were normalized to snoRNA202 expression [31 (link)]. Relative gene expression was determined using the 2-delta delta CT (2^-ΔΔC_T) analysis method.
Cells and tumor samples were also obtained for G6PD detection as described previously [5 (link)]. The relative CT method was employed to compare differences between samples. Fold-decrease/increase was determined relative to a blank control after normalization to the housekeeping gene GAPDH using the 2^-ΔΔCT method.

Total RNA was extracted using TRIzol^® Reagent (Invitrogen) as per the manufacturer’s instruction. RNA was quantified using the Nanodrop8000 spectrophotometer (Thermo Fisher Scientific). miRNA expression was determined using RNA from control and butyrate-treated (2.5 mM) CRC cells. cDNA was synthesized from 20–100 ng total RNA using microRNA- or RNU6B (endogenous control)-specific primers as specified by the TaqMan MicroRNA assay protocol (Thermo Fisher Scientific). Real-time RT–PCR was performed as specified by the TaqMan MicroRNA assay protocol in triplicate (Thermo Fisher Scientific), using miRNA-specific primers (assay IDs: miR-139-5p: 005364_mat and miR-542-3p: 001284). Thermocycling was performed using a Qiagen Rotorgene Q (Qiagen). Expression levels were calculated from Ct values using Qgene [67 (link)] and normalized against the endogenous small nuclear RNA gene, RNU6B (assay ID: 001093).

MiRNA reverse transcription was performed using 6,67 ng of total RNA, gene-specific primers (hsa-miR-215-5p; ID 000518, RNU48; ID 001006), and TaqMan™ MicroRNA Reverse Transcription Kit according to the TaqMan MicroRNA Assay protocol (Applied Biosystems, Foster City, CA, USA). MiRNA RT-qPCR was performed using TaqMan™ Universal Master Mix II, no UNG (Applied Biosystems) according to manufacturer’s recommendations. Gene expression reverse transcription was performed using 1000 ng of total RNA and Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s protocol. Gene expression RT-qPCR master mix was prepared using TaqMan™ Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) and gene specific primers TaqMan™ Gene Expression Assays (MYLK; ID Hs00364926_m1, ACVR2A; ID Hs00155658_m1, CBL; ID Hs01011446_m1, MSN; ID Hs00741306_mH, PMM1; ID Hs00160195_m1, GAPDH; ID Hs02758991_g1, PRKACB; ID Hs01086757_m1, CTNNBIP1; ID Hs00172016_m1, and CREB5; ID Hs00191719_m1), (Applied Biosystems, Foster City, CA, USA). RT-qPCR was performed using the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems).

Total RNA was isolated from human frozen tissues using the mirVana miRNA isolation kit (Ambion, Austin, Texas, USA) according to the manufacturer's instructions. The concentration and purity of RNA were spectrophotometrically determined by measuring the optical density (A260/280>2.0, A260/230>1.8) using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific Wilmington, DE, USA). cDNA was synthesized according to the TaqMan microRNA Assay protocol (Applied Biosystems) using 1 μg of total RNA as a template. qPCR analyses were conducted to quantify mRNA expression using SYBR Premix (Applied Biosystems) and GAPDH as an internal control. The sequences of the primers were as follows: FZD8 forward, 5′-GATGGGATTGCACGGTTTGG-3′, FZD8 reverse, 5′-ACCCGTATTTACGTGGGGTG-3′; GAPDHforward,5′-AATCCCATCACCATCTTCCA-3′, GAPDH reverse 5′-TGGACTCCACGACGTACTCA-3′. The TaqMan MicroRNA Assay kit (Applied Biosystems) was used to measure the levels of miR-375 after reverse transcribing with sequence-specific primers (Applied Biosystems), and U6 small nuclear RNA was used as an internal control. Real-time PCR was performed on an Applied Biosystems 7500 Sequence Detection System.