Total RNA from cells was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The cDNA was synthesized from 1 μg of total RNA using Moloney-murine-leukemia virus (M-MLV) Reverse Transcriptase (Enzynomics, Daejeon, Republic of KOREA) and oligo dT primer at 37°C for 1 hr. A one-twentieth aliquot of the cDNA was subjected to PCR amplification using gene-specific primers for FGF11 and farnesoid X receptor alpha (FXRα). PCR was carried out using the following forward and reverse primers: FGF11 F, 5′-CCA AGT CCC TTT GCC AGA AGC-3′; FGF11 R, 5′- CCA TGT AGT GAC CCA GCT TGG -3′; FXRα F, 5′- AGG GGA TGA GCT GTG TGT TG-3′; FXRα R, 5′-CCT GTA TAC ATA CAT TCA GCC AAC-3′. The cDNA was amplified by PCR and the PCR products were examined by electrophoresis on a 1% agarose gel. The RT-PCR bands were quantified relative to the β-actin control band. qRT-PCR was performed with TOPreal qPCR 2x PreMIX with SYBR Green (Enzynomics). The comparative CT method was used to calculate the relative gene expression levels with β-actin as an endogenous control gene.
Sybr green
Manufactured by Enzynomics
Sourced in United States
SYBR Green is a fluorescent dye that binds to double-stranded DNA. It is commonly used in real-time PCR (polymerase chain reaction) assays to detect and quantify specific DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus, by Takara Bio (17 mentions)
M mlv reverse transcriptase kit, by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Rneasy kit, by Qiagen (4 mentions)
Ex taq polymerase, by Takara Bio (3 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Lightcycler 480 real time pcr system, by Roche (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Cfx96, by Bio-Rad (3 mentions)
Topreal qpcr 2x premix, by Enzynomics (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Cfx system, by Bio-Rad (2 mentions)
Cfx instrument, by Bio-Rad (2 mentions)
Trizol isolation reagent, by Qiagen (2 mentions)
Revers transcription premix, by Elpis Biotech (2 mentions)
Rt200, by Enzynomics (2 mentions)
Taqman pcr master mix, by Thermo Fisher Scientific (2 mentions)
Topreal qpcr 2 premix, by Enzynomics (2 mentions)
54 protocols using sybr green
1
Quantification of FGF11 and FXRα Gene Expression
2
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The quantitative RT-PCR (qRT-PCR) was performed as previously described [14 (link)]. Briefly, total RNA was isolated from human lung cancer cells by using RNAiso Plus (TaKaRa, Otsu, Shiga Shiga 520–2193, Japan) according to the manufacturer’s instructions. Total RNA from each group of treated cells was converted to cDNA using a M-MLV reverse Transcriptase kit (Invitrogen, Carlsbad, USA) and SYBR green (Enzynomics, Seoul, Korea). The primers used for real-time PCR were Snail (forward) 5'-tcccgggcaatttaacaatg-3' and (reverse) 5'-tgggagacacatcggtcga-3' ; Twist (forward) 5'-cgggagtccgcagtctta-3' and (reverse) 5'-tgaatcttgctcagcttgtc-3' ; N-cadherin (forward) 5'-ctcctatgagtggaacaggaacg-3' and (reverse) 5'-ttggatcaatgtcataatcaagtgctgta-3' ; KAI1 (forward) 5'-gctcatgggcttgggct-3' and (reverse) 5'-gagctcagtcacgatgccgc-3' ; KITENIN (forward) 5’-cggaataaagacggcagagg-3’ and (reverse) 5’-tgctccgaggtgcctgtgat-3’ ; GAPDH (forward) 5’-atcaccatcttccaggagcga-3’ and (reverse) 5’-agttgtcatggatgaccttggc-3’ . qRT-PCR reactions and analyses were performed using CFX (Bio-Rad, Hercules, USA).
3
Quantitative Analysis of EGFR and GAPDH Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA (1 μg) from each cell was converted to cDNA using a M-MLV reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). Quantitative analysis of target and reference genes was performed in triplicate using SYBR green (Enzynomics, Seoul, Korea) on CFX (Bio-Rad, Hercules, CA, USA).
The primers used for the reaction were EGFR (forward) 5′-tggaaaacctgcagatcatc-3′ and (reverse) 5′-ttgctgagaaagtcactgct-3′; GAPDH (forward) 5′-atcaccatcttccaggagcga-3′ and (reverse) 5′-agttgtcatggatgaccttggc-3′. Results are obtained from five independent experiments.
The primers used for the reaction were EGFR (forward) 5′-tggaaaacctgcagatcatc-3′ and (reverse) 5′-ttgctgagaaagtcactgct-3′; GAPDH (forward) 5′-atcaccatcttccaggagcga-3′ and (reverse) 5′-agttgtcatggatgaccttggc-3′. Results are obtained from five independent experiments.
4
Isolation and Characterization of Neural Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used the RNeasy Kit (Qiagen, Hilden, Germany) to extract total RNA following the supplier’s instructions. Total RNA (1 μg) was reverse-transcribed into cDNA using the Omniscript RT Kit (Qiagen) following the manufacturer’s protocol. Total mRNA of human fetal brain (TaKaRa, Shiga, Japan) was used as a positive control to assess the expression of NSC-specific markers in derived NSCs. All RT-PCR mixtures contained Ex Taq Polymerase (TaKaRa); PCR was performed for 30 cycles for all markers. Imprinted gene expression levels were evaluated by quantitative real-time PCR using a 7500 Real-Time PCR System (Applied Biosystems, CA, USA) and SYBR Green (Enzynomics, Daejeon, Korea). All primer sequences are listed in Supplementary Table 1 .
5
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNA was extracted from cell lysates using the BioFACT Total RNA Prep Kit (BioFACT) and reverse transcribed with M‐MLV reverse transcriptase (TOYOBO) according to the manufacturer's instructions. The cDNA was mixed with SYBR Green (Enzynomics) and gene‐specific primers. All qPCR primers are listed in Table S2 , Supporting Information. All the qPCR analyses were performed in triplicate, and the relative mRNA expression levels were determined using the 2–ΔΔCt method.
6
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, total RNA was isolated from human lung cancer cells using RNAiso Plus (TaKaRa, Otsu, Shiga 520–2193, Japan) according to the manufacturer’s instructions. Total RNA (1 μg) from each group of treated cells was converted to cDNA using a M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, USA) and SYBR green (Enzynomics, Seoul, Korea). qRT-PCR reaction and analysis were performed using CFX (Bio-Rad, Hercules, USA).
7
Quantitative PCR Analysis of ADRB2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific). First-strand cDNA was synthesized using CellScript All-in-One 5X First Strand cDNA Synthesis Master Mix (CellSafe, Yongin, Korea) as described in the manufacturer’s protocol. cDNA templates diluted 50-fold in nuclease-free water were subjected to qPCR analysis using SYBR green (Enzynomics, Daejeon, Korea) and a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences were as follows: human ADRB2 forward (5′-TTGCTGGCACCCAATAGAAGC-3′) and reverse (5′-CAGACGCTCGAACTTGGCA-3′) and human GAPDH forward (5′-GTCTCCTCTGACTTCAACAGCG-3′) and reverse (5′-ACCACCCTGTTGCTGTAGCCAA-3′). The annealing temperature was 55 °C.
8
Comprehensive mRNA Extraction and Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For mRNA extraction, we used TRIzol and a total RNA extraction miniprep kit (#T2010S, NEB, USA). cDNA synthesis was performed using a Superscript III reverse transcription kit and a LunaScript RT Supermix kit (#E3010L, NEB, Ipswich, MA. USA). To validate the mRNA expression levels, semi- or real-time quantitative PCR was performed using SYBR green (#RT501M, Enzynomics, Daejeon, Korea) and primers (Supplementary Table S1 ) using the Stepone real-time PCR system (Applied Biosystems, Beverly, MA, USA).
To confirm the expression of the pluripotency markers, real-time quantitative-PCR was performed using TaqMan probes (SOX2 (#Hs00602736_s1, Thermo, Waltham, MA, USA), Nanog (#Hs02387400_g1,Thermo, Waltham, MA, USA) or Oct3/4 (#Hs04260367_Gh, Thermo, Waltham, MA, USA)) and pluripotency genes (SOX2, Nanog, Rex1, Oct3/4)
To confirm the expression of the pluripotency markers, real-time quantitative-PCR was performed using TaqMan probes (SOX2 (#Hs00602736_s1, Thermo, Waltham, MA, USA), Nanog (#Hs02387400_g1,Thermo, Waltham, MA, USA) or Oct3/4 (#Hs04260367_Gh, Thermo, Waltham, MA, USA)) and pluripotency genes (SOX2, Nanog, Rex1, Oct3/4)
9
Quantitative Real-Time PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The quantitative real-time PCR was performed as described previously (Nguyen et al., 2014 (link)). Briefly, the total RNA was isolated from Rv/C and Rv/δ cells and 1 μg of total RNA was converted to cDNA using M-MLV reverse Transcriptase kit (Invitrogen, USA). Real-time PCR analysis using a SYBR green (Enzynomics, Korea) was performed by CFX System (Bio-Rad, USA).
10
Quantitative Real-Time PCR Analysis of CSC221 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative RT-PCR (qRT-PCR) was conducted as already depicted3 (link). Total RNA was isolated from CSC221 cells using RNAiso Plus (TaKaRa, Otsu, Japan). 1 mg of RNA was converted to cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). qPCR was performed using SYBR Green (Enzynomics, Seoul, Korea)99 (link). Primers for real-time PCR are listed in Supplementary Table 2 . qRT-PCR reactions and analyses were performed on a CFX instrument (Bio-Rad, Hercules, CA, USA)100 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!