Lncprofiler qpcr array kit

For qPCR, 100 ng of total RNA was reverse-transcribed using the PrimeScript RT reagent Kit (Takara Bio Inc., Shiga, Japan) and KAPA SYBR FAST qPCR Kit Master Mix (2×) Universal (KAPA BIOSYSTEMS, Boston, MA, USA) according to the manufacturers’ recommendations, and MIQE guideline [52 (link)]. qPCR was performed using the Thermal Cycler Dice Real Time System (Takara Bio Inc.) or CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., CA, USA). All reactions were performed in triplicate. An lncRNA analysis was performed using the LncProfiler qPCR Array Kit (System Biosciences, Inc., Palo Alto, CA, USA), according to the manufacturer’s recommendations. For qPCR, 10 ng of total RNAs was reverse-transcribed using TaqMan probes (Thermo Fisher Scientific Inc.), according to the manufacturer’s recommendations. To perform ChIP-qPCR, genomic DNAs from formamide-fixed cells were analyzed using Chromatin Immunoprecipitation (ChIP) Kits (Takara Bio Inc.) according to the manufacture’s recommendations. The primers and antibodies are listed in S6 Table.

Total RNA was extracted from cells using TRIzol (Life Technologies) and treated with RNase-free DNase I (Qiagen, Valencia, CA). RNA concentration was measured using NanoDrop ND-2000 (Nano-Drop Technologies, Wilmington, DE). One microgram of RNA was reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (BIO-RAD Laboratories, Inc., Hercules, CA), and real-time quantitative RT-PCR was performed using a LightCycler 96 System (Roche Diagnostics, Mannheim, Germany) to detect BAP1, NEAT-1, NDM29, MER11C, SNHG4, β-actin, and U6 using SYBR green I (SYBR® Advantage® qPCR Premix, Clontech, Mountain View, CA). RT-PCR primer sequences are listed in Additional file 2: Table S2. PCR based expression profiling of lncRNA was performed using the LncProfiler qPCR Array Kit (System Biosciences, Mountain View, CA), according to the manufacturer’s instructions. RNA from BAP1 knock-down/out cells were treated with DNase I and 2 μg of DNase-treated RNA was reverse transcribed. Real-time PCR was performed (2X Maxima SYBR Green with Rox) and the cycle number at which the reaction crossed a threshold (CT) was determined for each gene. Raw CT values were normalized using a median CT value (ΔCT = CT_lncRNA- CT_median). For each lncRNA, the relative amount of each lncRNA between 2 sample sets A and B was described using the equation 2^-ΔΔCT, where ΔΔCT = ΔCT_A -ΔCT_B.

In this study, the 90 lncRNAs, potentially connected with cancer and well-annotated and registered in the lncRNA database (www.lncrnadb.org), were analyzed using the commercially available LncProfiler qPCR Array Kit (SBI).
Reverse transcription was performed according to the manufacturer’s protocol and was based on three steps: i) poly-A tailing; ii) annealing anchor dT adaptor; and iii) cDNA synthesis.
cDNA was used for the qRT-PCR reaction using LightCycler 480 SYBR Green I Master buffer (Roche) and lncRNA primers from Primer Plate (component of the LncProfiler qPCR Array Kit) according to the manufacturer’s protocol by the LightCycler 96 (Roche). All qRT-PCR data were analyzed by calculating the ΔC_t, normalized against mean expression of anti-nitric oxide synthase (NOS)2A+human accelerated region (HAR)1B+taurine upregulated gene (TUG)1, which were the most stable transcripts in all of the examined samples (healthy and cancer) with the lowest C_ts variation compared to the reference genes from the LncProfiler qPCR Array Kit (SBI). The fold-change of lncRNA expression was determined by equation 2^–ΔCt and compared to the appropriate group.