Glioma cell lines (U87-FGFR3 WT, SNB19.FGFR3 WT, and SNB19.F3T3) were transfected in triplicate with 25 pmol, 100 pmol, or 250 pm siRNA using Lipofectamine 2000 (ThermoFisher #11668027). Forty-eight hours following transfection, cells were lysed in either RIPA buffer with HALT protease and phosphatase inhibitors (ThermoFisher #78447) for protein lysates, or in QIAzol (Qiagen, #79306) for RNA. Total RNA was isolated using the miRNeasy Mini kit (Qiagen # 217004) and quantified using Nanodrop 2000c (check). cDNA was generated with the Superscript III First-Strand Synthesis Kit (ThermoFisher # 18080051) and oligo dT primers. Ten nanograms of cDNA was used per reaction for quantitative PCR with TaqMan Assays GAPDH (hs0275899_g1), FGFR3 (hs00997393_g1), and FGFR3-TACC3 (hs04396817_ft) and TaqMan Universal Master Mix II, no UNG (Applied Biosystems #4440040). Real-time PCR was performed in triplicate using the ABI7500_FAST PCR machine (Applied Biosystems) with the following PCR conditions: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. GAPDH was used as the internal reference gene, and relative quantitation was calculated using the 2−∆∆Ct method.
Nanodrop 2000c
Manufactured by Qiagen
Sourced in Germany
The Nanodrop 2000c is a spectrophotometer designed for the quantification and analysis of nucleic acids and proteins. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform measurements. The Nanodrop 2000c is capable of measuring the concentration and purity of DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Qiazol, by Qiagen (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Hiseq 2000 v4, by Illumina (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Rna stat 60, by Tel-Test (1 mentions)
Hiseq x, by Illumina (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
3 protocols using nanodrop 2000c
1
Quantifying FGFR3 and FGFR3-TACC3 in Glioma Cells
2
RNA-seq Analysis of Cellular Transcriptomes
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WT, JΔ, H4vΔ, H4vΔ JΔ, and Tet-treated or untreated TKO strains were grown in triplicate. About 50 million cells were collected, and total RNA was extracted using the RNA STAT-60 (Tel-Test) according to the manufacturer’s protocol, quantified on a NanoDrop2000c, and further cleaned using RNeasy kit (Qiagen). RNA-seq libraries were prepared from 500 ng of RNA samples using an oligo dT-based method with 15 cycles of PCR amplification with the Illumina TruSeq mRNA stranded kit (Illumina) and sequenced on the Illumina HiSeq 2000 v4 (50-bp single-end read). Reads were analyzed for transcription profile (Supplementary Table 5 ) and gene expression as above (Supplementary Table 4 ). Alignment report, correlation between replicates, and PCA are summarized in Supplementary Table 2 and Supplementary Figure 4 .
3
Z. officinale Genomic DNA Isolation
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Fresh Z. officinale leaves were collected from Laiwu City, Shandong Province, China, and were identified by Prof. Yulin Lin from the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences (CAMS). The voucher specimens were deposited in the herbarium of IMPLAD. Total genomic DNA was extracted from the clean sample leaves using a DNeasy Plant Mini Kit following standard protocol (Qiagen Co., Hilden, Germany) and the DNA concentration and quality were respectively assessed through Nanodrop 2000C spectrophotometry and electrophoresis in 1% (w/v) agarose gel. Approximately 1 μg genomic DNA was used for sequencing library construction. Paired-end libraries with insert sizes of 400 bp were prepared following Illumina’s standard genomic DNA library preparation procedure. Total DNA was sequenced in Illumina HiSeq X.
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