Total RNA was extracted using the TRI Reagent®isolation system (Sigma–Aldrich, St Louis, MO, United States). Moloney murine leukemia virus (M-MLV) reverse transcriptase and GoTaqPCR kits (Promega) were used for semiquantitative RT-PCR using primer sets as follows: HEY1, forward 5′-ATACGCCTGCATTTACCAGC-3′ and reverse 5′-TCAATTGACCACTCGCACAC-3′. Primer sets for NOTCH1, NOTCH2, and ACTB were published elsewhere (Hubmann et al., 2013 (link)). Real-time quantitative RT-PCR (qPCR) for NOTCH2 was performed with TaqMan®-probes (Hs01050717_m1) purchased from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, United States).
Tri reagent isolation system
Manufactured by Merck Group
Sourced in United States
The TRI Reagent isolation system is a laboratory product designed for the extraction and purification of RNA, DNA, and proteins from a variety of biological samples. It utilizes a single-step liquid-phase separation technique to isolate the target molecules efficiently.
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Lab products found in correlation
5 protocols using tri reagent isolation system
1
Quantitative Analysis of Notch Pathway Expression
2
MYC and NOTCH gene expression analysis
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Total RNA was extracted using the TRI Reagent® isolation system (Sigma-Aldrich, St Louis, MO, USA). M-MLV reverse transcriptase and GoTaq PCR kits (Promega, WI, USA) were used for semi quantitative RT-PCR. The MYC primer sequences used in this study read as follows: forward 5’-GAAAACAATGAAAAGGCCCC-3’ and reverse 5’-TTCCTTACGCACAAGAGTTC-3’. Primer sets for NOTCH1, NOTCH2, NOTCH3, FCER2, NR4A1, and ACTB were published elsewhere [32 (link)]. PCR bands were stained with GelRedTM (Biotium, Fremont, CA, USA) and visualized using the ChemiDocTM gel imaging system from Bio-Rad (Hercules, CA, USA).
3
Quantitative RT-PCR Analysis of Neuronal Transcripts
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RNA from neurons or NPCs was extracted using the TRI Reagent isolation system (Sigma-Aldrich) according to the manufacturer’s instructions. For quantitative RT-PCR (qRT-PCR), one microgram of RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega). Quantitative RT-PCR was performed in triplicate with custom-designed oligos using the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) using the Titan HotTaq EvaGreen qPCR Mix (BIOATLAS). cDNA was diluted 1:10, was amplified in a 16 µl reaction mixture containing 2 µl of diluted cDNA, 1× Titan HotTaq EvaGreen qPCR Mix (Bioatlas, Estonia), and 0.4 mM of each primer. Analysis of relative expression was performed using the ∆∆Ct method, using 18 S rRNA as housekeeping gene and CFX Manager software (Bio-Rad, USA). RNA was sequenced paired by GENEWIZ company (Germany). A list of primers is provided in the Supplementary Table 2 .
4
Quantitative RT-PCR Analysis of Gene Expression
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RNA was extracted using the TRI Reagent isolation system (Sigma-Aldrich) according to the manufacturer’s instructions. For quantitative RT-PCR (qRT-PCR), one microgram of RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega), thereafter qRT-PCR was performed in triplicate with custom-designed oligos (see Supplementary Dataset 4 ) using the CFX96 Real-Time PCR Detection System (Bio-Rad, USA). using the Titan HotTaq EvaGreen qPCR Mix (BIOATLAS). Obtained cDNA was diluted 1:10, and was amplified in a 16 µl reaction mixture containing 2 μl of diluted cDNA, 1× Titan HotTaq EvaGreen qPCR Mix (Bioatlas, Estonia), and 0.4 mM of each primer. Analysis of relative expression was performed using the ∆∆Ct method, using 18S rRNA as housekeeping gene and CFX Manager software (Bio-Rad, USA).
5
Gene Expression Quantification via qRT-PCR
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RNA was extracted using the TRI Reagent isolation system (Sigma-Aldrich) according to the manufacturer’s instructions. For quantitative RT-PCR (qRT-PCR) assays, 1 μg of RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega), thereafter qRT-PCR was performed in triplicate with custom designed oligos (Supplementary file 3 ) using the CFX96 Real-Time PCR Detection System (Bio-Rad, USA). using the Titan HotTaq EvaGreen qPCR Mix (BIOATLAS). Obtained cDNA was diluted 1:10 and was amplified in a 16 μl reaction mixture containing 2 μl of diluted cDNA, 1×Titan Hot Taq EvaGreen qPCR Mix (Bioatlas, Estonia) and 0.4 mM of each primer. Analysis of relative expression was performed using the ∆∆Ct method, using 18 S rRNA as housekeeping gene and CFX Manager software (Bio-Rad, USA).
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