Iscript cdna kit

Trizol (Invitrogen, USA) was utilized to extract total RNA from prostate biopsies with all operations performed following the manufacturer’s instructions. All RNA samples were digested with DNase I to remove genomic DNA. After cDNA synthesis through reverse transcriptions using Bio-Rad iScript cDNA Kit (Bio-Rad), PCR reactions were carried out using SYBR^® Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA). Thermal conditions were 95°C for 55s, and 40 cycles of 95°C for 22s and 60°C for 46s. Primers were: 5′-CCCAAGCTTGCGTTCTTTTC-3′ (sense) and 5′-CCGGAATTCTGACGGTAGTTTC-3′ (anti-sense) for lncRNA-SNHG16; 5′-AAGAAGCTGACGGGTCGCCTCATGC-3′ (sense) and 5′-TGAGAGGGACCAGAGCGTGGTG-3′ (anti-sense) for GLUT1; 5ʹ-GACCTCTATGCCAACACAGT-3ʹ (sense) and 5ʹ-AGTACTTGCGCTCAGGAGGA-3ʹ (anti-sense) for β-actin. 2^−ΔΔCT was employed to calculate the expressions.

Digested RNA samples were subjected to reverse transcription using Bio-Rad iScript cDNA Kit to prepare cDNA samples (Bio-Rad Laboratories, Inc.). With cDNA samples as template, qPCR reactions were prepared using SYBR-Green Master Mix (Bio-Rad Laboratories, Inc.). The expression levels of UASR1 and JAK2 mRNA were determined with GAPDH as the endogenous control. The expression levels of mature miR-375 were detected using All-in-One^™ miRNA qRT-PCR Detection Kit (GeneCopoeia) with U6 as the internal control. The sequences of the primers used were as follows: UASR1, forward, 5′-CCCTCCTCAAACACACATCC-3′ and reverse, 5′-TTAAGGAAATTAAAAATACC-3′; miR-375, forward, 5′-GGCTCTAGAGGGGACGAAGC-3′ and reverse, 5′-GGCAAGCTTTTTCCACACCTCAGCCTTG-3′; JAK2, forward, 5′-TCTATTTTATTATGGTTTCCCTTG-3′ and reverse, 5′-TTTTACTTATTTACCTCATTTCCC-3′; GAPDH, forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′; and U6, forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. PCR reactions were performed in triplicates and Cq values were processed using the 2^−ΔΔCq method (15 (link)).