Sybr green qpcr mix

The human glioma stem/progenitor cell line SU3 and nude mice expressing EGFP (NC-CB57/6J-EGFP) were prepared by our laboratory [4 (link), 5 (link)]. The remaining reagents were purchased from companies as follows: rat C6 glioma cell line (Shanghai Institutes for Biological Sciences), RFP transgenic kit (Genechem, Shanghai), γ-secretase inhibitors DAPT (GSI-IX) (Selleck), Dulbecco's Modified Eagle's Medium (Gibco), fetal bovine serum (Hyclone), Caspase—Glo 3/7 assay kit (Promega), Trizol solution (Invitrogen), reverse transcription kit (Fermentas), ECL chemiluminescence reagent, trypan blue, GAPDH antibody (Biyuntian, Shanghai), Notch-1, NICD, Bcl-2 and pAKT antibodies (Cell Signal), 2', 3'-cyclic nucleotide 3' phosphodiesterase (CNP) monoclonal antibody (Abcam), qPCR apparatus and SYBR Green qPCR Mix (BioRad), apoptosis kit (BD), flow cytometry instrument (Beckman), inverted fluorescence microscope (Carl Zeiss), linear accelerator (Siemens Primus), and in vivo fluorescence imaging system (Maestro EX, CRi).

The total RNA was isolated using RNAiso (Takara, Dalian, China). cDNA was subsequently reverse‐transcribed from mRNA by M‐MLV Reverse Transcriptase (Takara). The PCR included 40 cycles of amplification using the Stratagene Mx3000P system with SYBR Green qPCR Mix (BioRad, Hercules, California). Expression of target genes (2−^ΔΔCt) was normalized against GAPDH. The sequence of primer used in the qRT‐PCR:
Cyclin D1PF 5′‐ CAATGACCCCGCACGATTTC‐3′.
Cyclin D1 PR 5′‐ CATGGAGGGCGGATTGGAA‐3′.
Rb1 PF 5′‐TTGGATCACAGCGATACAAACTT ‐3′.
Rb1 PR 5′‐ AGCGCACGCCAATAAAGACAT ‐3′.
RBL1 PF 5′‐ ACCACCAAAGTTACCACGAAG ‐3′.
RBL1 PR 5′‐ CCCCAATCATCCGAAAATTACCC ‐3′.
P21 PF 5′‐ TGTCCGTCAGAACCCATGC ‐3′.
P21 PR 5′‐ TGTCCGTCAGAACCCATGC ‐3′.
GAPDH PF: 5′‐ TGTGGGCATCAATGGATTTGG‐3′.
GAPDH PR 5′‐ ACACCATGTATTCCGGGTCAAT‐3′.

The total RNA was isolated using RNAiso (Takara). cDNA was subsequently reverse‐transcribed from mRNA by M‐MLV Reverse Transcriptase (Takara). The PCR included 40 cycles of amplification using the Stratagene Mx3000P system with SYBR Green qPCR Mix (BioRad). Expression of target genes (2^−ΔΔCt) was normalized against GAPDH. The sequence of primer used in the qRT‐PCR: TP53BP2 PF 5′‐AGCTTGATCGCCTCTATAAGGA‐3′, PR 5′‐CCCTCAGCTCATTAACACGCT‐3′; GAPDH PF 5′‐TGTGGGCATCAATGGATTTGG‐3′, PR 5′‐ACACCATGTATTCCGGGTCAAT‐3′. E‐cadherin PF 5′‐ATTTTTCCCTCGACACCCGAT‐3′, PR 5′‐TCCCAGGCGTAGACCAAGA‐3′. Zeb1 PF: 5′‐CAGCTTGATACCTGTGAATGGG‐3′, PR 5′‐TATCTGTGGTCGTGTGGGACT‐3′. Snail PF 5′‐TCGGAAGCCTAACTACAGCGA‐3′, PR 5′‐AGATGAGCATTGGCAGCGAG‐3′. MMP2 PF 5′‐GATACCCCTTTGACGGTAAGGA‐3′, PR 5′‐CCTTCTCCCAAGGTCCATAGC‐3′.

The total RNA was isolated by using RNAiso plus (Takara, Japan). MiRNA was reverse‐transcribed to cDNA by using the miRNA‐specific stem‐loop reverse‐transcription primer (Ribobio, China). The amount of target gene expression (2^−ΔΔCt) was normalized via the endogenous small nuclear RNA U6 using miRNA‐specific primers (Ribobio). QRT‐PCR reaction conditions were obeyed by instructions of SYBR Green qPCR Mix (BioRad, Hercules, CA, USA).

RNAs from leaf samples were isolated using a Qiagen RNA extraction kit. A Takara Reverse Transcription Kit was employed for the synthesis of the first-strand cDNA. Equal amount of the total RNA for each of the sample was used to synthesize cDNA. qRT-PCR primers for each of the tested genes were designed (Supplementary Table S3). The wheat TaActin gene (GenBank accession AB181991.1) was employed as an inner reference gene [48 (link)]. The amplification efficiency was determined by a preliminary qRT-PCR assay using six 2-fold diluted cDNA samples (1:1, 1:2, 1:4, 1:8, 1:16, and 1:32). qRT-PCR reactions were performed using TransGen SYBR Green qPCR mix with a Bio-Rad CFX Manager instrument under the following conditions: 3 min at 95 °C; 40 cycles of 10 s at 95 °C, 10 s at 60 °C, and 10 s at 72 °C. Melting curves ranging from 60 °C to 94 °C were recorded by the instrument to evaluate the amplified product. The expression levels of target genes were relative to that of the reference TaActin gene in the same sample using the 2^-ΔCt method [49 (link),50 (link)]. The relative expression represents the ratio between the initial number of molecules of the target gene and that of TaActin. Therefore, the Y scales of the qRT-PCR results are comparable across genes and experiments.

RNA sample was isolated using RNAiso (Takara, Otsu, Japan). SYBR‐Green qPCR Mix (Bio‐Rad) was used to amplify template DNA. The abundance of H19, miR‐520a‐3p and LIMK1 was analyzed using the formula of 2^−ΔΔCt and normalized to U6 small nuclear RNA or glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Relevant primers were listed as follows: H19, Forward (F), 5’‐GATGACAGGTGTGGTCAACG‐3’, Reverse (R), 5’‐CAGACATGAGCTGGGTAGCA‐3’; miR‐520a‐3p, F, 5’‐ACACTCCAGCTGGGAAAGTGCTTCCC‐3’, R, 5’‐CTCAACTGGTGTCGTGGA‐3’; LIMK1, F, 5’‐ATGAGGTTGACGCTACTTTGTTG‐3’, R, 5’‐CTACACTCGCAGCACCTGAA‐3’; U6, F, 5’‐CTCGCTTCGGCAGCACA‐3’, R, 5’‐AACGCTTCACGAATTTGCGT‐3’; GAPDH, F, 5’‐TGGGTGTGAACCACGAGAA‐3’, R, 5’‐GGCATGGACTGTGGTCATGA‐3’.