Microrna specific stem loop primers

Plasma levels of target microRNAs were further tested by quantitative reverse transcription-polymerase chain reaction using the TaqMan method [TaqMan miRNA Reverse Transcription Kit and microRNA-specific stem-loop primers (Applied Bio Systems)] in a small-scale RT reaction for primers sequence, see Supplementary file. The RT reaction consists of a fixed volume of 1.67 µL total RNA solution of each sample as input, 1.387 µL of H₂O, 0.5 µL of 10× reverse-transcription buffer, 0.063 µL of RNase-inhibitor (20 U/µL), 0.05 µL of 100 mM deoxynucleotide triphosphates (dNTPs) with deoxythymidine triphosphate (dTTP), and 0.33 µL of MultiScribe Reverse-Transcriptase (50 U/µL). The 5 µL reactions were incubated in an Applied Biosystems 7500 Real Time PCR System in a 96-well plate at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min, and held at 4 °C. All RT reactions were run in duplicate. The levels of the synthetic spiked-in RNAs were measured in each of the samples and showed no signs of abnormal RNA loss during the extraction process of any of the samples.

Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse-transcribed complementary DNA was synthesized with random primers or microRNA specific stem-loop primers (Applied Biosystems, Foster City, CA, USA). Subsequently, the cDNA was subjected to real-time PCR on a 7500 real-time PCR system (Applied Biosystems). The 2^−∆∆Ct method was used to calculate the relative expression levels of the genes of interest. GAPDH and U6 were used as internal controls. The primer sequences used were as follows: GSN sense: 5′-CAGACAGCCCCTGCCAGCACCC-3′, antisense: 5′-GAGTTCAGTGCACCAGCCTTAGGC-3′; GAPDH sense: 5′-AGCCTCCCGCTTCGCTCTCT-3′, antisense: 5′-GCGCCCAATACGACCAAATCCGT-3′; miR-200a sense: 5′-GGCGTAACACTGTCTGGTAA-3′, antisense: 5′-CGTATCCAGTGCGTGTCGTG-3′; U6 sense: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, antisense: 5′-CGCTTCACGAATTTGCGTGTCAT-3′.

Reverse transcription reactions are performed using the TaqMan microRNA Reverse Transcription Kit and microRNA-specific stem-loop primers (Lot No.4366597, Applied BioSystems, Inc.) Real-time PCR reactions were performed in 20 μL reaction volumes using 10 μL TaqMan 2× Universal PCR Master Mix with 1 μL miRNA-specific primer, and 1.33 μL diluted RT product per reaction. All assays were carried out on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) using the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The comparative threshold cycle (ΔC_t) method was used to measure the expression level of miR-30a-3p relative to the expression of an internal control (U6 small nuclear RNA). All the primers are listed in Supplementary Table 4.