Transcript first strand synthesis supermix

RNA was isolated from kidneys tissue using TRIzol Reagent (Invitrogen, Cat. 15596-018), reverse transcription was performed using Transcript First Strand Synthesis Supermix (TransGen Biotech, Cat. AT301), according to the manufacturer’s instructions. All qRT-PCRs were performed using a 7900 Fast Real-Time PCR System (Applied Biosystems), and SYBR Green PCR Master mix was purchased from Applied Biosystems (Applied Biosystems, Cat. 43-676-59). Each sample was analyzed in triplicate or greater replicates. Relative quantification was derived from the difference in the cycle threshold (Ct) between the target gene and GAPDH (ΔCt) and compared with control cell lines using the equation RQ=2-ΔΔCt. Error bars represented the standard deviation (SD), and the significance of any differences was calculated using a two-tailed, unpaired t-test.

RNA was isolated from cell lines or patient samples using TRIzol Reagent
(Invitrogen, 15596-018) according to the manufacturer's protocol. To
obtain cDNA, reverse transcription was performed using Transcript First
Strand Synthesis Supermix (TransGen Biotech, Beijing, China AT301) according
to the manufacturer's instructions, using 1 μg of
RNA as a template. All qRT-PCRs were performed using a 7500 Fast Real-Time
PCR System (Applied Biosystems, Carlsbad, CA, USA), and all qRT-PCR reagents
and consumables were purchased from Applied Biosystems. For each reaction,
1 μl of RT product was added to
10 μl of 2X SYBR Green Gene Expression PCR Master Mix
and 1 μl of pre-designed and synthesized forward and
reverse primer/probe mix. Each sample was analyzed in triplicate.
Relative quantification (RQ) was derived from the difference in the cycle
threshold (Ct) between the target gene and GAPDH (ΔCt) compared with
control cell lines using the equation
RQ=2^−ΔΔCt. Error bars represent the
standard deviation (SD), and the significance of differences was calculated
using a one-tailed, unpaired t-test. The sequences of the primers
are listed in Supplementary Table 1. The
patients were dichotomized on the basis of the mean value of Bcl-3 mRNA
expression, and their survival curves were later analyzed.

Total RNA including miRNAs was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. To obtain cDNA, Transcript First-Strand Synthesis Supermix (TransGen Biotech, AT301) was used to generate cDNA following the manufacturer's instructions using 1 μg RNA as the template. Actin was used as an internal control gene. For miRNA, 500 ng total RNA was reverse-transcribed into cDNA using a specific miRNA stem loop primer. miR-U6 was used as an internal control miRNA. qRT-PCR reagents purchased from TaKaRa and a 7900 Fast Real-Time PCR System (Applied Biosystems) were used for quantitative real-time reverse transcription (qRT-PCR). All samples were analyzed in triplicate. Error bars represent the standard deviation (SD), and statistical significance was calculated using a one-tailed, unpaired t-test. The relative quantification (RQ) was derived from the difference in cycle threshold (Ct) between the target gene and internal control (actin or miR-U6) compared to control cell lines using the formula RQ = 2^-ΔΔCt. The mRNA and miRNA levels were assessed by SYBR Green–based quantitative real-time PCR with gene-specific primers (Supplementary Table S1).

Total miRNAs were isolated from cultured cells or surgically resected from fresh NSCLC tissues with a mirVana miRNA Isolation Kit (Ambion) in accordance with the manufacturer's instructions. Total RNAs were isolated from cell lines with TRIzol Reagent (Invitrogen, 15596-018) as described in the manufacturer's protocols. To obtain cDNA, we performed a reverse transcription reaction with Transcript First Strand Synthesis Supermix (TransGen Biotech, AT301) according to the manufacturer's instructions, using 1 μg total RNAs as the template. For miRNA, 0.5 μg total RNA from each sample was reverse-transcribed to cDNA by means of specific miRNA stem loop primers. All quantitative real-time reverse transcription PCR (qRT-PCR) was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems), and all qRT-PCR reagents and consumables were purchased from Applied Biosystems and TaKaRa. The mRNA and miRNA levels were quantitatively assessed by SYBR Green-based qRT-PCR with gene-specific primers. The sequences of primers are listed in Supplementary Table S1. GAPDH and U6 were used as internal normalization controls.