Revertaid first strand dna synthesis rt kit

Total RNA was isolated using TRIzol reagent (Invitrogen, USA). After quantification, total cDNA was synthesized using the RevertAid First-Strand DNA Synthesis (RT) Kit (Fermentas, Canada). Then, quantitative real-time PCR was performed with the SYBR Premix Ex Taq system (TaKaRa, Japan) and the Bio-Rad CFX96TM Real-Time PCR System (Bio-Rad, USA). The endogenous control was β-actin, and the primer sequences are shown in Table 1. The 2^−ΔΔCt method was used to determine the relative expression level of target genes.Table 1

The primers of β-actin and nine randomly selected lncRNAs

LncRNA	Primer
RP11-823E8.3	F:5ʹ-TTCTTTAGGCGGTGTGTGGC-3ʹR:5ʹ-TCGGTCACTAAAGCCCAGTC-3’
THAP9-AS1	F:5ʹ-ACTGGCTGCTATGGAAAAAGT-3ʹR:5ʹ-TCCCTTCCCCTCCTGTCTGC-3’
MIR22HG	F:5ʹ-AAGAACTGTTGCCCTCTGCC-3ʹR:5ʹ-AATCTGGGCAAAGGCTCTCC-3’
SNHG1	F:5ʹ-CTTATTGGGCTCCTGCTCGC-3ʹR:5ʹ-ACCAGTAAGCTCTTGTGGGC-3’
LRRC75A-AS1	F:5ʹ-AGAGTGCTGAAGACGGGGTA-3ʹR:5ʹ-TCAAAACCTCATGGCAGGCT-3’
GAS5-001	F:5ʹ-TGCAGGCAGACCTGTTATCC-3ʹR:5ʹ-CCATGAGACTCCATCAGGCA-3’
GAS5-016	F:5ʹ-GCCGAGTCACCCGAGTAAG-3ʹR:5ʹ-CTACCACCGACAGCCTTTCA-3’
XLOC_314741-TCONS_00422456	F:5ʹ-TTCTTCCTGTGTGTTGGGGC-3ʹR:5ʹ-GTAGTCGGGCACTGGTTTCA-3’
XLOC_158512-TCONS_00206520	F:5ʹ-TTCATCAATGTGTTCGTGAAGC-3ʹR:5ʹ-ACGGTTGTGCTTCTCTGTCC-3’
β-actin	F:5ʹ-AGTTGCGTTACACCCTTTCTTG-3ʹR:5ʹ-CACCTTCACCGTTCCAGTTTT-3’

Abbreviations: F, forward primer; R, reverse primer.

We used the TRIzol reagent (Invitrogen) according to the manufacturer's instructions to extract the total RNA. We then reverse transcribed the RNA to cDNA with the RevertAid First Strand DNA Synthesis (RT) Kit (Fermentas). Then, quantitative real‐time PCR was performed with SYBR® Premix Ex Taq™ II (Takara) using an ABI Prism 7900 sequence detection system (Applied Biosystems) as follows: 1 cycle of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s. Melt curve analysis was performed to confirm the specificity of the amplified product. β‐Actin was used as an endogenous control. Relative expression of the target genes was determined using the 2^−ΔΔCt method. The primers are listed in Appendix Table A1.