Sds relative quantification software version 2

To validate the microarray results in the study, relative quantitative real-time PCR was performed on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA) using SYBR Green I on nine differentially expressed miRNAs. Complementary DNA (cDNA) was generated by reverse transcription of 200 ng of total RNA using miScript RT II Kit (Qiagen, Hilden, Germany). Briefly, 200 ng of total RNA containing miRNAs was mixed with miScript 4 µL HiSpec Buffer, 2 µL nucleic mix, 2 µL miScript Reverse Transcriptase mix and RNase-free water to a final volume of 20 µL. Following the reverse transcription reaction, the cDNA was diluted 1:10 and then mixed with 10 µL QuantiTect SYBR Green PCR Master Mix, 2 µL miScript Universal Primer, 2 µL miScript Primer Assay for the selected nine miRNAs namely miR-328-5p, miR-4750-5p, miR-210-5p, miR-423-3p, miR-143-3p, miR-564, miR-770-5p, miR-874-5p, miR-93-5p and RNU6B as endogenous control and RNase-free water to a final volume of 20 µL. The primer sequences used in the study are shown in Additional file 1: Table S1. Reactions were assembled with the QIAgility automated pipetting system (Qiagen, Hilden, Germany). All PCR data were analyzed with SDS Relative Quantification Software version 2.3 (Applied Biosystems, Foster City, USA).

Total RNA was extracted from the collected fresh-frozen tissue specimens using the standard Trizol method; miRNA expression was measured with the microRNA assay (Applied Biosystems Inc, US). The expression analysis is described briefly as follows. First, 25 ng of total RNA were reverse-transcribed to cDNA using the TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems Inc., US). The reverse transcription (RT) was completed sequentially under the following conditions: incubation at 16°C for 30 minute, 42°C for 30 minute and 85°C for 5 minute. After RT, quantitative polymerase chain reaction (qPCR) was performed in the 7900 HT-Fast real-time PCR system (Applied Biosystems Inc., US) following the protocol of denaturing at 95°C for 10 minute, and 40 cycles of denaturing at 95°C for 15 second and annealing and elongation at 60°C for 1 minute. The qPCR results were analyzed with the SDS Relative Quantification Software version 2.1 (Applied BioSystems Inc., US). Small RNA RNU6 was utilized as an endogenous control to normalize the quantity of cDNA used for analysis of miR-195 expression. All samples were analyzed in triplicate, and the measurement was repeated if the coefficient of variation was greater than 5%. The expression level of miR-195 was calculated based on the formula 2^−△△Ct, where ∆Ct = Ct _(miR-195)–Ct_(RNU6).

TaqMan based qPCR reactions were performed using the ABI PRISM 7900HT Sequence Detector System (Applied BioSystems, Foster City, CA, USA) together with a commercially available predesigned microRNA assay, primers, and probes as shown in Tables 1 and 2. Taqman microRNA assay for small nuclear RNA U6 (snU6) was used to normalize the miRNAs amplifications, whereas the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the endogenous control for mRNA gene expression. MicroRNA and mRNA data were collected and subsequently analyzed using the SDS Relative Quantification Software version 2.1 (Applied BioSystems), in which the comparative C_T method (2^−ΔΔCT) for relative quantification was employed [44] (link). Results in the figures are expressed as the expression of fold change relative to mean value of the control group, which was equal to 1.

Total RNA was reverse transcribed using the TaqMan miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Applied BioSystems) in a small-scale RT reaction (comprised of 0.19 ml of H₂O, 1.5 ml of 10X Reverse-Transcription Buffer, 0.15 ml of 100 mM deoxyribonucleotide triphosphates, 1.0 ml of Multiscribe Reverse-Transcriptase (50 U/ml), and 5.0 ml of input RNA (20 ng/ml); components other than the input RNA were prepared as a larger volume master mix), using a Tetrad2 Peltier Thermal Cycler (Bio-Rad, Alpha Technologies Ltd, Wicklow, Ireland) at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. For miRNAs and snoRNA, 4.0 ml of RT product was combined with 16.0 ml of PCR assay reagents (comprised of 5.0 ml of H₂O, 10.0 ml of TaqMan 2X Universal PCR Master Mix, No AmpErase UNG, and 1.0 ml of TaqMan miRNA Assay) to generate a PCR of 20.0 μl of total volume. Real-time PCR was carried out on an Applied BioSystems 7900HT thermocycler at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed with SDS Relative Quantification Software version 2.2.2 (Applied BioSystems.), with the automatic Ct setting for assigning baseline and threshold for Ct determination.

TaqMan low-density arrays (TLDA; human microRNA Cards A v2.1 & B v2.0, Applied Biosystems, Darmstadt, Germany) were used for measuring the expression of 667 human miRNAs from miRBase version v.10 with an Applied Biosystems 7900HT. Raw data were exported using SDS Relative Quantification Software version 2.2.2 (Applied Biosystems) with automatic baseline and threshold settings.

To detect plasma levels of lncRNA, 2 μL of the cDNA product was used as template in 10 μL reaction containing 5 μL of TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster, CA), 1 μL of specific primer (Supplementary Table 1), 2 μL of RNase-free water. qRT-PCR was performed with 7900HT real-time PCR system (Applied Biosystems, Foster, CA) at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Triplicate measurements were obtained for each sample on a 384-well plate. Data were analyzed with SDS Relative Quantification Software version 2.2.2 (Applied Biosystems, Foster, CA), with the automatic Ct setting for assigning baseline and threshold for Ct determination. The relative expression level of each individual lncRNA after normalization to cel-miR-39 was calculated using the 2^−ΔΔCt method.