One step sybr primescript rt pcr kit

Total RNA was extracted from human tissue samples and HA, U87 and U251 cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA). RNA quality and concentration were measured via 260/280 nm absorbance with Nanodrop Spectrophotometer (ND-100, Thermo, Waltham, MA). SNHG20, FOXK1 mRNA, GAPDH mRNA concentration were detected using One-Step SYBR PrimeScript RT-PCR Kit (TakaraBio, Inc., Japan) in 7500 Fast RT-PCR System (Applied Biosystems, USA). The relative quantification (2^-△△Ct) was calculated after the expression levels were normalized using GAPDH as endogenous control.

SFN mRNA was measured by real-time PCR. Total RNA was extracted from the cells using RNeasy spin columns (Qiagen, Hilden, Germany). The extracted total RNA was then quantitatively measured using a One Step SYBR PrimeScript RT-PCR Kit (Takara, Kyoto, Japan) and the ABI PRISM 7700 PCR thermal cycler. The β-actin gene (ACTB) was used as the internal standard. The PCR primers with the following sequences were used: SFN-F, 5′-TGACGACAAGAAGCGCATCAT; SFN-R, 5′-GTAGTGGAAGACGGAAAAGTTCA; ACTB-F 5′-CACCATTGGCAATGAGCGGTTC; ACTB-R, 5′-AGGTCTTTGCGGATGTCCACGT.

Total RNA was extracted from plasma, brain tissue, and cells using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA). RNA concentrations were detected, and the quality was determined at 260/280 nm absorbance using an Ultra‐micro UV Spectrophotometer (N50 Touch; Implen, Germany). One Step SYBR PrimeScript RT‐PCR Kit (Takara Bio, Inc., Japan) was applied to determine KQNQ1OT1 expression. TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to reverse transcribe cDNA from miRNA. Quantification reactions were implemented using TaqMan Universal Master Mix II with TaqMan microRNA assays for mmu‐miR‐200a and U6 (Applied Biosystems, Foster City, CA, USA) via the ABI 7,500 Fast Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA). The fold change was equivalent to the relative expression normalized to endogenous control (2^−ΔΔCt). The primers sequences were as follows: hsa‐KCNQ1OT1: forward 5'‐TGCAGAAGACAGGACACTGG‐3' and reverse 5'‐CTTTGGTGGGAAAGGACAGA‐3'; hsa‐GAPDH: forward 5'‐ TGCACCACCAACTGCTTAGC‐3' and reverse 5'‐GGCATGCACTGTGGTCATGAG‐3'; mmu‐KCNQ1OT1: forward 5'‐GCCAAGCCTGATGACCTAAACTC‐3' and reverse 5'‐ ACTCCCCATCCTCAATTCCTCTA‐3'; and mmu‐GAPDH: forward 5'‐ CCTCGTCCCGTAGACAAAATG‐3' and reverse 5'‐TGAGGTCAATGAAGGGGTCGT‐3'.

Total RNA was extracted from cell lines using TRIzol (Invitrogen) following the manufacturer’s protocol. One hundred nanograms of each triplicate sample was used in real-time RT-PCR using One-Step SYBR PrimeScript RT-PCR kit (Takara, Cat#RR086A) following the manufacturer’s protocols. Thermal cycle program for DDR2 primers: 50 °C 2 min, 95 °C 20 s, 40 cycles of 95 °C 3 s, 60 °C 30 s, and 72 °C 5 s, and Col1a1 primers: 42 °C 5 min, 94 °C 5 min, 30 cycles of 94 °C 12 s, 60 °C 8 s, 72 °C 8 s, both followed by a melt curve of 65 °C to 95 °C in 0.4 °C increments. Experiments were carried out in triplicate for each data point, and relative expression was determined using 2^−ΔΔCτ method.

Gene	Forward	Reverse
Human DDR2	5′-TCA CCC AGA CCC ATG AAT AC-3′	5′-GGG AAG GAA ATG GCA TTA GG-3′
Human Col1a1	5′-TCT GCG ACA ACG GCA AGG TG-3′	5′-GAC GCC GGT GGT TTC TTG GT-3′
Human β-actin	5′-ATC CTC ACC CTG AAG TAC CC-3′	5′-TAG AAG GTG TGG TGC CAG AT-3′