Trizoltmplus rna isolation reagents

Total RNA was extracted from GC tissues and cells using TRIzol^TMPlus RNA Isolation Reagents (Invitrogen, Waltham, MA, USA). A Reverse Transcription Kit (Takara, Otsu, Japan) was used for reverse transcription. qRT-PCR was performed on an ABI 7500HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 10 min, 40 cycles at 95°C for 10 s, 60°C for 20 s and 72°C for 34 s. GAPDH was used as the internal control. The mRNA expression level was calculated using the 2^−ΔΔCt method. The primer sequences were HCG18, forward: 5′-ATCCTGCCAATAGATGCTGCTCAC-3, reverse: 5′-AGCCACCTTGGTCTCCAGTCTC-3′; GAPDH, forward: 5′-TGACGTGCCGCCTGGAGAAAC-3, reverse: 5′-CCGGCATCGAAGGTGGAAGAG-3′.

TRIzol^TM Plus RNA Isolation Reagents (Invitrogen, Carlsbad, CA, USA) was utilized to extract total RNA from tissues and cells. RNA reverse transcription was performed using reverse transcription kit (Takara, Otsu, Japan). qRT-PCR was carried out using SYBR Premix Ex Taq (Takara) on ABI 7500HT Fast Real-Time PCR System (Applied Biosystems, CA, USA). The amplification program was as follows: 95°C for 3 min, 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 20 s. 2^−ΔΔCt method was utilized to calculate the mRNA expression level. The primer sequences were as follows: SNHG14, forward: 5ʹ-AAGGTGGGGTAAGCACACTG-3ʹ and reverse: 5ʹ-CCGAACAAGTGTCCAGGAAT-3ʹ; miR-93-5p, forward: 5ʹ-TCTACAGTGCACGTGTCTCCAG-3ʹ, reverse: 5ʹ-ACCTGCGTAGGTAGTTTCATGT-3ʹ; ZBTB7A, forward: 5ʹ-ATCTGCGAGAAGGTCATCCA-3ʹ, reverse: 5ʹ-CAGCAGCTGTCGCACTGGTA-3ʹ; GAPDH: forward: 5ʹ-GACGGCCGCATCTTCTTGT-3ʹ and reverse: 5ʹ-CACACCGACCTTCACCATTTT-3ʹ; U6, forward:5ʹ-GCTTCGGCAGCACATATACTAAAAT-3ʹ and reverse: 5ʹ-CGCTTCACGAATTTG CGTGTCAT −3ʹ.

Total RNA was extracted from cells using TRIzol^TMPlus RNA Isolation Reagents (Invitrogen). The reverse transcription kit (Takara, Otsu, Japan) was applied for RNA reverse transcription. qRT-PCR was performed on ABI 7500HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following reaction conditions: 95 °C for 3 min, 40 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for 20 s. The mRNA expression level was calculated according to the 2^–ΔΔCt method. U6 or β-actin was used as the internal reference of miR-150 or IGF2BP1, respectively (Table 1).