Genes were validated by RT-qPCR assays. The RT-qPCR reactions were performed on a Roche LightCycler Instrument 1.5, using a LightCycler® FastStart DNA Master PLUS SYBR-Green I kit (Roche cat. no. 03515885001; Roche Diagnostics Australia Pty Limited, Castle Hill, Australia). Briefly, the reactions had a 15 μl volume: 7.5 μl Master mix, 0.1 μl forward primer and reverse primer, 1 μl cDNA sample and 6.3 μl ddH2O were prepared. Each sample was run in triplicate. The RT-qPCR program was set to 95°C for 5 min and subsequently 45 cycles of 95°C for 10 s, 55–60°C for 35 s and 72°C for 40 s. At the end of each program, a melting curve analysis was performed. In addition, the data were automatically analyzed by the system and an amplification plot was generated for each cDNA sample at the end of each qPCR run. The reference gene was β-actin. The primers of each gene used are shown in Table 1 .
Lightcycler instrument 1
Manufactured by Roche
Sourced in Germany
The LightCycler Instrument 1.5 is a real-time PCR system designed for nucleic acid amplification and detection. It features a thermal cycler and optical detection system for quantitative analysis of DNA and RNA samples.
Automatically generated - may contain errors
5 protocols using lightcycler instrument 1
1
Validated Gene Expression Analysis by RT-qPCR
2
Quantitative Real-Time PCR for WNT5A Expression
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Quantitative PCR (QRT-PCR) was carried out on the Light Cycler Instrument 1.5 (Roche Diagnostics, Germany) with SYBR Premix Ex Taq (TAKARA, RR420A). The PCR conditions were prepared as per the instructions of the manufacturer and all samples were studied in duplicate. The specificity of amplification of the products was confirmed by melting curve analyses and agarose gel electrophoresis. The PCR program was as follows: initial denaturation at 95 °C for 7 min; amplification segment of 5 s at 95 °C, 10 s at 59 °C, and 10 s at 72 °C for 45 cycles; and melting curve segment of 15 s at 60 °C for 1 cycle. WNT5A relative expression levels were normalized to 3 reference genes (β-actin, CypA, and ABL).
3
Quantitative Assessment of KIT D816V Allele Burden
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Quantitative assessment of the KIT D816V expressed allele burden (EAB) at RNA-level was performed by allele-specific RT-qPCR. Two PCR assays were designed for amplification of total KIT transcripts and KIT D816V mutated transcripts. KIT D816V EAB was calculated as ratio between mutant KIT D816V and total KIT transcripts. Limit of detection reveals a sensitivity of 0.01–0.1%. PCR was performed using the universal “mastermix” (LightCycler® FastStart PLUS set, Roche Diagnostics, Mannheim, Germany) and specific primer and probes on a LightCycler® instrument 1.5 (Roche Diagnostics, Mannheim, Germany) in a final volume of 20 μL with 2 μL cDNA or plasmid product (500 nm primer; 250 nm probes). Thermocycling conditions were as follows: 95 °C (10 min), 45 cycles: 95 °C (1 s), 60 °C (10 s), and 72 °C (26 s) [13 (link)].
4
Extracting and Analyzing Total RNA from Oral Cancer Tissues
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The total RNA was extracted from oral cancerous tissues, as well as their adjacent noncancerous tissue using TRIzol (Invitrogen, Carlsbad, CA, USA) and the commercial protocol of the manufacturer as described [21 (link)]. Before further real-time qRT-PCR PCR analysis, each cDNA pool was prepared at −20°C. For real-time PCR assays, specific oligonucleotide primer pairs were purchased from Roche Universal ProbeLibrary. The reactions of real-time qRT-PCR were analyzed using the Roche LightCycler Instrument 1.5 with a LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Cat. 03 515 885 001, Castle Hill, Australia). The fold change of the expression of the target gene relative to the internal control gene GAPDH in each sample was calculated using the following formula: 2−ΔΔCt where ΔCt = Cttarget gene − Ctinternal control and ΔΔCt = ΔCttest sample − ΔCtcontrol sample.
5
Quantitative gene expression analysis
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Real-time PCR was performed using a LightCycler Instrument 1.5 (Roche) and LightCycler® FastStart DNA MasterPLUS SYBR Green I kit (cat. no. 03 515 885 001; Roche, Castle Hill, Australia). Briefly, 10 µL of sample, comprised of 2 µL of Master Mix, 2 µL of 0.75 µM forward and reverse primers, and 6 µl of cDNA sample, were run. Each sample was run in triplicate using the following program: 95 °C for 10 min, 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 10 s. At the end of the program, melting curve analysis was performed. At the end of each RT-PCR run, the data were automatically analyzed by the system, and an amplification plot was generated for each cDNA sample. From each of these plots, the LightCycler3 Data analysis software automatically calculated the Ct (crossing point) value, which denotes the turning point corresponds to the first maximum of the second derivative curve, used to indicate the beginning of exponential amplification. The mRNA fold expression or repression of the target gene relative to the internal control gene GAPDH in each sample was then calculated using the 2−△△Ct method, based on the following formula:
This method was used to analyze the relative gene mRNA expression through real-time quantitative PCR.
This method was used to analyze the relative gene mRNA expression through real-time quantitative PCR.
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