Total RNA of glioma cell lines were extracted using the EasyPure Total RNA reagent (Bioman, Taiwan, ROC) according to the manufacturer's protocol. Briefly, cells were harvested and lysed in EasyPure Total RNA reagent. After the addition of chloroform followed by centrifugation, the aqueous phase were transferred into a new tube for RNA recovery by precipitation with isopropyl alcohol. cDNA were prepared using oligo dT, and MMLV Reverse transcriptase (Epicentre Biotechnologies, USA). The normal brain cDNA was purchased from Origene Technologies (Rockville, MD, USA).
Normal brain cdna
Manufactured by OriGene
Sourced in United States
Normal brain cDNA is a collection of complementary DNA molecules derived from messenger RNA (mRNA) extracted from healthy brain tissue. It serves as a representative sample of the genes expressed in the normal brain.
Automatically generated - may contain errors
Lab products found in correlation
Mmlv reverse transcriptase, by Illumina (2 mentions)
Eco real time pcr system, by Illumina (1 mentions)
Oligo dt primer, by Illumina (1 mentions)
U87mg, by American Type Culture Collection (1 mentions)
U118mg, by American Type Culture Collection (1 mentions)
Gbm8401 glioma cell line, by RIKEN BioResource Center (1 mentions)
4 protocols using normal brain cdna
1
Extraction and Reverse Transcription of Glioma Cell RNA
2
Quantitative Real-Time PCR Analysis of PLOD3 Expression
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We performed total mRNA extraction, reverse transcription and quantitative RT-PCR according to the manufacturer’s protocol. Normal brain cDNA was purchased from Origene Technologies (Rockville, MD, USA). The reverse transcripts were amplified and quantified using an Illumina ECO™ Real-Time PCR system. The relative quantitative gene expression was normalized to GAPDH as an internal control and calculated using the 2−ΔΔCt method. The primer pairs used were as follows: PLOD3 forward 5′-GACCCGGTCAACCCAGAGA-3′ and reverse 5′-CTCCACCAACTGTTCGAGCC-3′ [65 (link)]; GADPH forward 5′-CTTCATTGACCTCAACTAC-3′ and reverse 5′-GCCATCCACAGTCTTCTG-3′.
3
Quantitative RT-PCR Analysis of SGO2 Expression
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Total RNA was extracted using EasyPure Total RNA reagent (Bioman, Taipei, Taiwan) according to the manufacturer’s protocol. For cDNA synthesis, 1.0 μg RNA was reverse transcribed into cDNA using Oligo dT primer with MMLV Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI, USA). Normal brain cDNA was purchased from Origene Technologies (Rockville, MD, USA).
Gene expression was quantified using quantitative RT-PCR (qRT-PCR) and performed in an illumina ECOTM Real-Time PCR system. Amplifications were performed using an IQ2 fast qPCR system with ROX (Bio-genesis Technology Inc., Taipei, Taiwan). Relative quantitative gene expression against an internal control, GAPDH, was performed using the 2−ΔΔCt method22 (link). The primer pairs used were SGO2 forward, 5′-ATGTGGTGCATGGCCTAAAAA-3′ and reverse, 5′-GGGGTACATATTGGTGATCTGC-3′ and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-ATGGTGGTGAAGACGCCAGT-3′.
Gene expression was quantified using quantitative RT-PCR (qRT-PCR) and performed in an illumina ECOTM Real-Time PCR system. Amplifications were performed using an IQ2 fast qPCR system with ROX (Bio-genesis Technology Inc., Taipei, Taiwan). Relative quantitative gene expression against an internal control, GAPDH, was performed using the 2−ΔΔCt method22 (link). The primer pairs used were SGO2 forward, 5′-ATGTGGTGCATGGCCTAAAAA-3′ and reverse, 5′-GGGGTACATATTGGTGATCTGC-3′ and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-ATGGTGGTGAAGACGCCAGT-3′.
4
Quantification of DPY19L1 Expression in Glioma Cells
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We extracted total mRNA via TRIzol™ Reagent (Thermo Fisher Scienti c, Wal-tham, WA, USA) according to the manufacturer's protocol. Oligo dT primer with MMLV Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI, USA) was applied for cDNA synthesis. We purchased normal brain cDNA from Origene Technologies (Rockville, MD, USA). Ampli cation and quanti cation of DPY19L1 expression were achieved by a StepOne™ Real-Time PCR System (Thermo Fisher Scienti c, USA). We used the 2 -ΔΔCt method to compare relative quantitative gene expression with GAPDH as an internal control. The primer pairs included presented below: DPY19L1 forward 5′-ACACCACCTCTCCGTGAAAGCT-3' and reverse 5′-GCAGAGTGCAATCAA-GCTTCCTC-3'; GADPH forward 5′-CTTCATTGACCTCAACTAC-3′ and reverse 5′-GCCATCCACAGTCTTCTG-3′.
Cell Culture LN229, U118MG and U87MG cell lines were commercially available from American Type Culture Collection (ATCC), and we also purchased GBM8401 glioma cell line from Bioresource Collection and Research Center (BCRC number 60163, Hsinchu, Taiwan). LN229 and GBM8401 cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) which comprises 2% fetal bovine serum (FBS), penicillin, and streptomycin, and U87MG, U118MG, and LNZ308 cells were cultured in DMEM consisting of 10% FBS, penicillin, and streptomycin. Cells above were incubated in 37°C and 5% CO2 condition.
Cell Culture LN229, U118MG and U87MG cell lines were commercially available from American Type Culture Collection (ATCC), and we also purchased GBM8401 glioma cell line from Bioresource Collection and Research Center (BCRC number 60163, Hsinchu, Taiwan). LN229 and GBM8401 cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) which comprises 2% fetal bovine serum (FBS), penicillin, and streptomycin, and U87MG, U118MG, and LNZ308 cells were cultured in DMEM consisting of 10% FBS, penicillin, and streptomycin. Cells above were incubated in 37°C and 5% CO2 condition.
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